Proc. Natl. Acad. Sci. USAVol. 89, pp. 6993-6997, August 1992Biochemistry

Binding of transition metals by apolipoprotein A-I-containingplasma lipoproteins: Inhibition of oxidation of lowdensity lipoproteins

(high density lipoproteins/transferrin/iron/ceruloplasmin/copper)

STEVEN T. KUNITAKE, MICHAEL R. JARVIS, ROBERT L. HAMILTON, AND JOHN P. KANE

Cardiovascular Research Institute and the Departments of Medicine and Anatomy, University of California, San Francisco, CA 94143-0130

Communicated by Richard J. Havel, April 1, 1992

ABSTRACT We have found transition metals tightlybound to apolipoprotein A-I-containing lipoproteins [Lp(A-I)]isolated by selected affinity immunosorption from humanserum. Prominent among the metal ions detected were iron andcopper. By immunoblotting the proteins of Lp(A-I), we de-tected both transferrin and ceruloplasmin. The transferrin-containing Lp(A-I) particles, isolated by selected aflmity im-munosorption against transferrin, were larger (mean diameterof 14.2 nm) and had a higher protein content than most highdensity lipoproteins (HDL). Ultracentrifugally isolated HDLwere found to contain much less transferrin, whereas trans-ferrin was found associated with apolipoprotein A-I from the>1.21-g/ml ultracentrifugal fraction. This suggests that thecomplex is not recovered in the classic HDL density intervalbecause of its very high density. HDL inhibit copper-catalyzedoxidation of low density lipoproteins (LDL) in vitro. We havefound that immunoisolated Lp(A-I) are an order of magnitudemore effective in inhibiting the oxidation of LDL than ultra-centrifugally isolated HDL, on the basis of protein mass. Whenthe Lp(A-I) particles containing transferrin and ceruloplasminwere removed from the bulk of Lp(A-I), inhibition of the invitro oxidation of LDL was significantly decreased.

High density lipoproteins (HDL) include a number of discretelipid-protein complexes (1-7). They are thought to perform awide variety of functions (8-15) in addition to their involve-ment with cholesterol transport (16). Diversity in functioncould be a reflection of the differing protein componentsresiding on individual HDL species.Loss of apolipoproteins associated with HDL during iso-

lation by sequential ultracentrifugation has been well docu-mented (17-20). Other proteins not considered to be integralto HDL but which associate partially with HDL underphysiological conditions might also become lost from theHDL density fraction during ultracentrifugation due either todissociation or by having a particle density outside the HDLdensity range. Loss of such elements could seriously limitdetection of special functions of HDL.

In contrast, isolation of apolipoprotein (apo) A-I-containing lipoproteins [Lp(A-I)] by selected affinity immu-nosorption (SAIS) appears to retain apolipoproteins other-wise lost from HDL during sequential ultracentrifugation(21-23). Lecithin-cholesterol acyltransferase (LCAT) andcholesteryl ester transfer protein (CETP) remain bound toimmunoisolated Lp(A-I) and not to ultracentrifugally isolatedHDL (24).

In this study we found that Lp(A-I) preferentially bindtransition metals-in particular, iron and copper. This asso-ciation appears to be attributable to the presence of Lp(A-I)containing transferrin and ceruloplasmin. We have found that

the oxidation of low density lipoproteins (LDL) by copper, invitro, is inhibited by Lp(A-I) particles containing transferrinor ceruloplasmin.

METHODSReagents. We obtained goat antisera against transferrin,

ceruloplasmin, and apo A-II from International Immunology(Murrieta, CA). Anti-apo A-I and apo A-I immunodiffusionplates were from Tago. Transferrin, ceruloplasmin, and a2-macroglobulin were from Boehringer Mannheim; cyanogenbromide-activated Sepharose from Pharmacia; a choline as-say from Wako (Osaka); protein standard from Pierce; andmicrowell plates from Costar. Other materials were fromSigma.Serum Samples. Venous blood was drawn from fasting

normolipidemic men and women. The blood was allowed toclot for 1 hr at 40C. The serum was recovered by centrifu-gation at 1000 x g for 30 min at 40C. Gentamicin sulfate,benzamidine, and phenylmethanesulfonyl fluoride (10 ug/ml, 0.3 mg/ml, and 10 ,ug/ml, respectively) were added to theserum stored at 40C.

Isolation of Lipoproteins. Lp(A-I) were isolated by SAISusing antibodies against apo A-I, as described (21, 22, 25).Column buffers containing 5 mM Tris and 0.15 M NaCl (pH7.4, TBS) or 0.2M acetic acid and 0.15 M NaCl (pH 3.0) wereprepared with deionized, double-distilled water and weredevoid of EDTA and sodium azide.

Lp(A-I) containing transferrin were isolated from totalLp(A-I) by a second step of SAIS with antibodies againsthuman transferrin. Ceruloplasmin complexes were removedfrom Lp(A-I) by a third separate SAIS step. A sham columnwas prepared, exactly as the immunosorption column againstapo A-I, with antibodies isolated from nonimmune goatserum. The eluate from the sham column was compared withthat from an anti-apo A-I column run in an identical manner.Transferrin was measured in equal volumes of eluate from theanti-apo A-I and sham columns.LDL (1.019-1.063 g/ml) and HDL (1.063-1.21 g/ml) were

isolated by repeated sequential ultracentrifugation (26). Thelipoproteins were dialyzed against TBS containing 0.04%EDTA and 0.05% sodium azide.

Characterization of Lipoproteins. The metal ion contents oflipoprotein samples were determined by atomic absorptionspectroscopy (27) in the Microanalytical Laboratory of theUniversity of California at Berkeley with a Perkin-Elmermodel 2380 atomic absorption spectrophotometer. All sam-ples were dialyzed against TBS, and their metal ion contents

Abbreviations: apo, apolipoprotein; CETP, cholesteryl ester transferprotein; HDL, high density lipoprotein(s); LCAT, lecithin-cholesterol acyltransferase; LDL, low density lipoprotein(s); Lp(A-I), apo A-I-containing lipoprotein(s); SAIS, selected affinity immu-nosorption; TBARS, thiobarbituric acid-reactive substances.

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FIG. 1. Immunodetection of transferrin. Ten micrograms ofLp(A-I) (lane A), an equivalent volume ofeluate from a sham column(lane B), and 1.0 ,g of transferrin (lane C) were subjected toSDS/PAGE. After transfer to nitrocellulose the samples were im-munoblotted with antibodies against human transferrin. Transferrin(arrow) was detected in Lp(A-I) but barely discernable in the shameluate. The high molecular weight material detected on the immu-noblots appears to be trace amounts of IgG present in the samples.

were calculated after subtracting the contents of the dialy-sate.The protein content of lipoproteins was determined by the

method ofLowry et al. (28), cholesterol and cholesteryl esterby an enzymatic method (29), phospholipid as choline (Wako)or as lipid-associated phosphorus (30), and triacylglycerolsby their glycerol content (31).

Competitive ELISA techniques were developed for mea-surement of apo A-I and transferrin. Linear responses wereobtained for apo A-I between 0.5 and 40 ng and for transferrinbetween 1.5 and 100 ng.Diameters of the lipoproteins were determined from elec-

tron micrographs by a computer-based method (32). Particlesizes were confirmed by nondenaturing gradient gel electro-phoresis (5) and gel filtration with a Pharmacia Sepharose 12column. The proteins were analyzed by electrophoresis in0.1% sodium dodecyl sulfate on a 5-25% polyacrylamidegradient gel (SDS/PAGE) (25). Gels were stained withCoomassie blue R-250 or immunoblotted on nitrocellulose(33).

Oxidation ofLDL. LDL (10 mg/ml) isolated in the presenceof0.04% EDTA were dialyzed against 150 mM NaCl/40 mMphosphate, pH 7.0/0.01% EDTA and stored at 4°C. The LDLwere diluted to a final concentration of 100 ,ug/ml in 150 mMNaCl/40 mM phosphate, pH 7.4/5 ,uM CUSO4. In separatetubes increasing amounts ofHDL or Lp(A-I) were added tothe LDL. HDL and Lp(A-I) were dialyzed in TBS devoid ofEDTA; additions were made such that equal volumes ofHDL/dialysate were added to each LDL incubation. As a

control, EDTA at a final concentration of 200 ,uM was addedto one tube of diluted LDL. The mixtures were incubated for90 min at 37°C. We found that production of thiobarbituric

FIG. 2. Transferrin immunoblots of HDL. One microgram oftransferrin (lane A), 10 ,ug of immunoisolated Lp(A-I) (lane B), and20 ,Ag of centrifugally isolated HDL (lane C) were subjected toSDS/PAGE. After transfer, nitrocellulose sheets were immunoblot-ted with antibodies against human transferrin. Transferrin could bedetected in both of the HDL samples; however, the centrifugallyisolated HDL contained much less transferrin.

acid-reactive substances (TBARS) reached a plateau withinthis time (data not shown). Further incubation caused thedensity of the LDL to increase to >1.065 g/ml, interferingwith the reisolation of the LDL. Oxidation was terminatedwith EDTA and butylated hydroxytoluene added to finalconcentrations of 200 AuM and 50 ,uM, respectively. The LDLwere analyzed directly or after isolation by ultracentrifuga-tion of the incubation mixture at a density of 1.065 g/ml.LDL oxidation was measured by the production ofTBARS

(34). We obtained similar results whether we measuredTBARS directly from the incubate or after LDL recovery.Seven of eight HDL samples did not appear to generatesignificant TBARS within 90 min. The unmodified free amino

Table 1. Distribution of plasma transferrin bound to Lp(A-I)Transferrin,

mg/mg of apo A-I % transferrinSubject Plasma Lp(A-I) bound to Lp(A-I)

1 1.82 0.0040 0.142 1.68 0.0042 0.153 1.62 0.0038 0.15

FIG. 3. Electron micrograph of transferrin-containing Lp(A-I).Transferrin-containing Lp(A-I) were negatively stained with phos-photungstate, and electron micrographs were taken (A). The lipo-proteins had an average particle diameter of 14.2 + 2.3 nm. TotalLp(A-I) are shown for comparison (B). (x 180,000.)

A B C

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Table 2. Percent chemical composition of transferrin-containing Lp(A-I)Free Cholesteryl Triacyl-

Protein Phospholipid cholesterol ester glycerolTransferrin-containing Lp(A-I) 89 ± 7 8.8 ± 1.0* 1.1 ± 0.1 1.1 ± 0.2 NDLp(A-I) pool 65.5 ± 3.9 19.5 ± 1.2 1.3 ± 0.3 11.1 ± 0.3 4.2 ± 0.5

Percent chemical composition is derived from a pool of transferrin-containing HDL isolated from three different subjects.ND, not determined.*Determined by an enzymatic assay measuring choline only.

groups on LDL were measured by reaction with trinitroben-zenesulfonic acid (35), and the extent of degradation of theapo B-100 was monitored by SDS/PAGE (36).

RESULTSWe have found several transition metals bound to Lp(A-I).Among those detected in samples from five individuals werecobalt [42 + 39 ng/mg of Lp(A-I) protein], iron (212 ± 293),molybdenum (28 ± 26), chromium (14 22), manganese (7 ±16), nickel (26 ± 30), and copper (120 96). Magnesium (19

32) and aluminum (141 179) were also detected.To determine whether the binding of transition metals to

HDL was due to specific binding proteins, we analyzedLp(A-I) and identified a subfraction that contained tightlybound transferrin by immunoblotting proteins separated bySDS/PAGE (Fig. 1). The transferrin contents of threeLp(A-I) samples were found to be 0.15 ± 0.01% of plasmatransferrin (Table 1).

In contrast to Lp(A-I), the eluate from a sham columncontained only trace amounts of transferrin (Fig. 1). Trans-ferrin was found associated with ultracentrifugally isolatedHDL but at a very low level (Fig. 2). Transferrin wasdemonstrable by immunoblotting when we examined the apoA-I-containing particles isolated by immunosorption from the>1.21-g/ml density fraction of human serum (data notshown).

Transferrin-containing Lp(A-I) were isolated by SAIS us-ing affinity-purified antibodies against human transferrin.The particles obtained were distinctly larger than the bulk ofLp(A-I) (14.2 + 2.3-nm mean diameter; Fig. 3). The size wasconfirmed by gel filtration chromatography and by nondena-turing gradient gel electrophoresis (data not shown). Inelectron micrographs, the particles do not appear to possesstranslucent lipid-rich cores. They contain 89% protein, 9%phospholipid, and minute amounts of nonpolar lipid (Table2). Transferrin and apo A-I were the major proteins. No apoA-II was detected on immunoblots (Fig. 4).

Lp(A-I) were also found to contain ceruloplasmin byimmunoblotting (data not shown). Only trace amounts ofceruloplasmin were detected in the eluate from the shamcolumn, and the amount of ceruloplasmin in ultracentrifu-

A B C D E

FIG. 4. Electrophoresis of transfemrn-contaiming Lp(A-I). After

SDS/PAGE, the separated proteins were stained with Coomassie

Blue r-250 (lane A) or immunoblotted with antisera to transferrin

(lane C), apo, A-I (lane D), and apo A-II (lane E). Markers corre-

sponding to 14.4, 21.5, 31.0, 42.7, 66.2, and 97.4 kDa are shown for

comparison (lane B).

gally isolated HDL appeared to be greatly reduced comparedwith Lp(A-I) (data not shown). The quantity of the cerulo-plasmin lipoprotein complex that could be isolated wasinsufficient for measurement of its lipid and protein constit-uents.The iron and copper contents of several HDL samples

isolated by immunosorption or ultracentrifugation were de-termined by atomic absorption spectroscopy (Table 3). Theconcentration of bound iron [average, 34 ng/mg of Lp(A-I)protein] and copper [36 ng/mg of Lp(A-I) protein] variedabout 2-fold in these subjects. Copper and iron were asso-ciated with ultracentrifugally isolated HDL from subjects 1and 2, but their concentrations were dramatically reduced.After removal of transferrin- and ceruloplasmin-containingcomplexes from Lp(A-I) of subjects 1 and 2, iron and copperwere no longer detectable, suggesting that their presence inHDL was attributable to the transferrin and ceruloplasmincomplexes (Table 3).HDL can inhibit Cu2+-catalyzed oxidation ofLDL in vitro

(37, 38). Immunoisolated Lp(A-I) were almost an order ofmagnitude more effective at inhibiting the production ofTBARS in LDL than an equal mass of ultracentrifugallyisolated HDL from the same individual (Fig. 5). Lp(A-I) alsoappeared to protect the free amino groups of apo B, asdetermined by reactivity with trinitrobenzenesulfonic acid(Fig. 6), and to inhibit the degradation of apo B-100 (data notshown). Removal of transferrin- and ceruloplasmin-contain-ing lipoproteins from Lp(A-I) reduced the ability of theremaining Lp(A-I) to inhibit the oxidation ofLDL by almostone-half (Fig. 7). In preliminary experiments, we couldinhibit the oxidation of LDL with transferrin or ceruloplas-min alone, but only at concentrations that were =2 orders ofmagnitude higher than were present in Lp(A-I) that producedthe same degree of inhibition (data not shown).

DISCUSSIONWe found that transition metals bind to Lp(A-I). These metalions are all known ligands of transferrin or ceruloplasmin.The subpopulations of Lp(A-I) that bind these ions appear tocontain transferrin and ceruloplasmin and to inhibit theoxidation of LDL, in vitro.

Table 3. Iron and copper contents of HDL samplesFe* Cu*

Immunoisolated Lp(A-I)Subject 1 26 24.4Subject 2 23.5 39.1Subject 3 52 44

Centrifugally isolated HDLSubject 1 1.8 0.0Subject 2 0.8 0.7

Immunoisolated Lp(A-I) after removalof transferrin and ceruloplasmin

Subject 1Subject 2

-, Not detectable (<0.003 ng).*ng/mg of HDL protein.

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FIG. 5. Inhibition of the oxidation of LDL by HDL. The pro-duction of TBARS during Cu2+-catalyzed oxidation of LDL wasmeasured as a function of the amount of centrifugally isolated HDL(*) or Lp(A-I) (8) added to the LDL before incubation at 370C for90 min. The TBARS produced per mg of apo B protein are expressedrelative to that amount produced in the absence of inhibitors. Eachpoint represents the mean of two values. These results are repre-sentative of five experiments.

Isolation of Lp(A-I) by selected affinity immunosorptionyields lipoprotein particles that retain more proteins than dolipoproteins isolated by sequential ultracentrifugation (21-23). An increased content of apo A-IV (39), CETP (24), andLCAT (24) has been reported when HDL were isolated byminimally perturbing methods. In addition, previously un-documented proteins have been coisolated with Lp(A-I)obtained by immunoaffinity chromatography. James et al.(40) have detected at least six unidentified protein spots bytwo-dimensional gel electrophoresis of Lp(A-I) proteins. Bytransblotting of two-dimensional gels and sequence analysis,we have identified a number ofplasma proteins in associationwith Lp(A-I) (41). As with these selected plasma proteins,transferrin appears to have significant affinity for Lp(A-I).Three observations indicate that the association of trans-

ferrin with Lp(A-I) is not artifactual. (i) Transferrin is alsodetectable in HDL isolated by ultracentrifugation, an inde-pendent method ofisolation. Additionally, we detected trans-

0)

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Lp(A-I) Concentration(gg)

FIG. 6. Inhibition of the modification of free amino groups inLDL by Lp(A-I). The free amino groups on apo B were measured asa function of Lp(A-I) added to the incubation mixture. The LDL andLp(A-I) mixtures were incubated for 90 min at 37C in the presenceof 5 ,uM Cu2+. After reisolation, unmodified amino groups on apo Bwere measured by reaction with trinitrobenzenesulfonate and ex-pressed in values relative to the free amino groups found in nativeLDL. Each point represents the mean of two values. These resultsare representative of three experiments.

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0.0 * * * *0 20 40 60 80 100 120

Lp(A-i) Protein Concentration (gg/mL)

FIG. 7. Effect of transferrin/ceruloplasmin-containing Lp(A-I)on the inhibition of the oxidation of LDL. The production ofTBARSduring the Cu2+-catalyzed oxidation of LDL was determined as afunction of the amount of Lp(A-I) (E) and Lp(A-I) devoid oftransferrin and ceruloplasmin (*) mixed with the LDL prior toincubation. The TBARS produced per mg of apo B protein areexpressed relative to the amount produced in the absence of inhib-itors (means oftwo values). These results are representative of threeexperiments.

ferrin-apo A-I complexes in the >1.21-g/ml centrifugal frac-tion. (ii) The failure to recover significant quantities oftransferrin from sham columns indicates that transferrin isnot directly bound to the anti-apo A-I columns. (iii) Immu-nosorption columns prepared with antibody against transfer-rin also retain apo A-I, implying that the two proteins resideon the same complex.

Oxidation of LDL is thought to be a key element in theatherosclerotic process (42). HDL can inhibit the Cu2+-catalyzed oxidation of LDL in vitro (37, 38). We have foundthat Lp(A-I) are more effective in the inhibition of LDLoxidation than ultracentrifugally isolated HDL and that asignificant part of this in vitro inhibition can be attributed tothe quantitatively minor subfractions of transferrin- andceruloplasmin-containing Lp(A-I). Ohta et al. (38) have foundthat particles with apo A-I but without apo A-II possess agreater inhibitory capacity than particles containing both apoA-I and apo A-II. Our finding that apo A-II cannot bedetected in transferrin-containing Lp(A-I) particles is con-sistent with this finding. Klimov et al. (43) suggested thatLCAT is capable of inhibiting the oxidation of LDL (43).Perhaps the LCAT-containing Lp(A-I) along with the metal-binding Lp(A-I) can account for the inhibition of the oxida-tion ofLDL that we observe with immunoisolated Lp(A-I) invitro.

Parthasarathy et al. (44) have found that HDL do not blockthe ultimate oxidation of LDL in a 24-hr incubation; how-ever, the HDL did appear to block the uptake ofthe oxidizedLDL by macrophages. This finding, coupled with the previ-ously stated results, suggests that HDL may have two effectsupon LDL oxidation.The importance of this in vitro phenomenon to the oxida-

tion ofLDL in vivo is unknown. Non-HDL-associated trans-ferrin is present in much greater abundance in plasma than inHDL. Even though transferrin alone has been shown toinhibit the oxidation ofLDL (45), it appears that the levels oftransferrin and ceruloplasmin needed to inhibit the oxidationof LDL are -2 orders of magnitude more than that com-plexed with Lp(A-I). Also, we found that adding Lp(A-I) tothe incubation mixture did not significantly change the bulkconcentration of free copper (data not shown). Perhapstransferrin-containing Lp(A-I) may function by interacting insome specific way with the substrate LDL. Transferrin is apotent inhibitor of transition metal-catalyzed reactions,which lead to the formation of free radicals in biological

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systems. The coupling of transferrin with a lipoprotein maythus allow the localization of this metal-sequestering activityin selected tissue sites. The existence of a parallel lipoproteincomplex containing ceruloplasmin may reflect coupled ac-tivity between ceruloplasmin and transferrin. The ferroxi-dase activity of ceruloplasmin allows the oxidation of Fe(II)to Fe(III) without the production of oxygen radicals and hasbeen shown to inhibit iron-mediated oxidation of fatty acids.Even though the subfraction of Lp(A-I) containing trans-

ferrin is small compared with plasma levels of either trans-ferrin or apo A-I, it may nevertheless have significance inlipoprotein metabolism. The interaction between transferrinand the transferrin receptor has been well documented (46).It is thus possible that the transferrin receptors may providea pathway for the endocytosis of at least one subfraction ofHDL. In addition, transferrin has been reported to stimulatecell replication. Perhaps the presence of transferrin on HDLmight explain the stimulation of replication of endothelialcells, which has been described with HDL (8). In any case,the association of metal-binding proteins with lipoproteinsmay confer special biochemical properties on the complexwith respect to localization ofthese functions at specific sites.

We wish to express our appreciation to Marion Merrill-DowPharmaceuticals, Inc. This work was supported by National Insti-tutes of Health Grants HL31210 and HL14237 (ArteriosclerosisSCOR) and grants from the California State Tobacco Related DiseaseProgram, the Wine Institute, and Merck and Co., Inc.

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