Low-density lipoprotein oxidation, antioxidants, and atherosclerosis

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Low-density lipoprotein oxidation, antioxidants, and atherosclerosis Powered By Docstoc
					Clinical Chemistry         42:4
498-506        (1996)




      Low-density lipoprotein oxidation, antioxidants,
        and atherosclerosis: a clinical biochemistry
                        perspective
                                                 ISHWARLAL              JIALAL*      and SRIDEvI                     DEVARAJ




Cardiovascular disease is the leading cause of mortality in                                taken up by the scavenger      receptor  mechanism,   resulting   in
westernized     populations.   An increased concentration      of                          cholesterol  accumulation   and subsequent   foam cell formation,
plasma low-density lipoprotein (LDL) cholesterol consti-                                   since the scavenger receptor is not regulated by the cholesterol
tutes a major risk factor for atherosclerosis.   Several lines of                          content within the cell [1].’
evidence support a role for oxidatively modified LDL in
atherosclerosis    and for its in vivo existence. Antioxidants                                                         Mechanisms of LDL Oxidation
have been shown to decrease atherosclerotic        lesion forma-                           LDL oxidation                is generally                   believed        to occur              mainly       in the
tion in animal models and decrease LDL oxidation; the                                      intima       of the artery,              in microdomains                 sequestered               from      antioxi-
evaluation of LDL oxidation in vivo is therefore very im-                                  dants.       Several        lines of evidence                       observed           by different            groups
portant. However, there is a paucity of methods for direct                                 over       the     years     support              a role        for    Ox-LDL                  in atherogenesis
measurement       of LDL oxidation. Of the direct methods                                  [2-6].       LDL       can be oxidatively                    modified           in a cell-free             system by
currently   available, the preferred ones seem to be the                                   transition         metals         such      as iron          and      copper          and      by all the        major
measurement       of F2-isoprostanes,   autoantibodies    to epi-                          cells of the arterial                 wall such            as endothelial             cells,     smooth        muscle
topes on oxidized LDL, and the assessment of antioxidant                                   cells, and             monocyte-macrophages.      Physiologically relevant
status. Of the indirect measures, the most uniformly ac-                                   mechanisms              underlying   LDL oxidation in vivo are yet to be
cepted procedure is examining the oxidative susceptibility                                 established.           Various studies implicate superoxide                                        anion as one
of isolated LDL by monitoring conjugated diene formation.                                  agent that             promotes  oxidation   of LDL lipids,                                        mediated  by
                                                                                           smooth           muscle          cells     and        phagocytes               [7].     A well-understood
INDEXING     ThRMS:     cholesterol      cardiovascular
                                      #{149}                  disease      a-toco-
                                                                        #{149}             pathway           is the    membrane-associated                          NADPH                  oxidase       of acti-
           ascorbate #{149}
pherol #{149}              13-carotene #{149} conjugated        dienes #{149}
                                                                            apoli-         vated phagocytes.                  Activated           human           neutrophils               and monocytes
poprotein    B-lOO . fatty acids #{149}      thiobarbituric        acid-reactive           oxidize          LDL       via        a pathway              that      is inhibited               by      superoxide
               prostaglandins
substances #{149}                    isoprostanes
                                  #{149}                                                   dismutase and metal chelators [8, 9]. Thiols autooxidize in the
                                                                                           presence of metal ions, forming thiyl radicals and superoxide,
An increased concentration            of plasma low-density        lipoprotein             which promote LDL oxidation [10]. It has been proposed that
(LDL) cholesterol       constitutes     a major risk factor for atheroscle-                arterial         smooth      muscle           cells        reduce      disulfides              to thiol      intracel-
rosis. Clinical, epidemiological,         and genetic studies convincingly                 lularly and export thiol to the extracellular medium; the thiol
demonstrate     that LDL promotes atherosclerosis.             However, the                then autooxidizes, forming a species that can promote oxidation.
precise mechanism(s)        by which LDL promotes the development                          LDL          oxidation           by      thiols       in     a cell-free              system       supports         this
of the early fatty-streak        lesion still remains to be elucidated.                    hypothesis.    Certain cellular enzymes, such as 15-lipoxygenase,
Uptake of cholesterol         by the classical LDL receptor pathway                        that convert     polyunsaturated    fatty acids (PUFAs) into lipid
cannot result in appreciable           cholesterol   accumulation       because            hydroperoxides      may also oxidize LDL [11]. Soybean    lipoxyge-
the LDL receptor          is subject to feedback         inhibition      by the
intracellular   cholesterol     content [1]. However,       modified forms
of LDL such as acetyl LDL or oxidized LDL (Ox-LDL)                            are
                                                                                                ‘Nonstandard abbreviations: Ox-LDL, oxidized LDL; PUFA, polyunsatu-
                                                                                           rated fatty acid; AAPH, 2,2’-az.obis(2-amidinopropane); apo, apolipoprotein; CD,
                                                                                           con(ugated diene; MDA, malondialdehyde; HNE, 4-hydroxynonenal; MM-LDL,
    Departments     of Internal Medicine and Pathology and Center for Human                minimally modified LDL; MCP-1, monocyte chemotactic protein-I; M-CSF,
Nutrition, University of Texas Southwestern Medical Center, Dallas, TX 7523 5-             macrophage colony-stimulating factor; IL-i, interleukin-1; EDRF, endothelium-
9052.                                                                                      derived relaxation factor; BHT, butylated hydroxytoluene; PBS, phosphate’
      Author for correspondence. Fax 214-590-2785.                                         buffered saline; TBARS, thiobarbituric acid-reactive substances; GC, gas chroma-
    Received September 18, 1995; accepted January 12, 1996.                                tography; MS, mass spectrometry; and PGF2, prostaglandin F2.



                                                                                     498
                                                              Clinical Chemistiy    42, No. 4, 1996                                                                  499




nase and phospholipase
LDL oxidation
protein and mRNA
                               A2 have also been shown to stimulate
                       in the absence of cells [11]. 15-Lipoxygenase
                            have been found in atherosclerotic
[12], although some groups question the role of 15-lipoxygenase
in LDL oxidation. The heme protein, myeloperoxidase,
creted by activated phagocytes,
acting as a physiological
                                          may also oxidize lipoproteins
                                 catalyst. The products of myeloperox-
                                                                         lesions

                                                                              se-
                                                                               by
                                                                                         Unsaturatei

                                                                                        acid
                                                                                                         latty




                                                                                                                 AAAJ
                                                                                                                       S
                                                                                                                           I   -             abstraction




idase action, hypochlorous acid and tyrosyl radical, promote                                                                   Molecular rearrangement
lipoprotein oxidation [13, 14]. Nitric oxide and peroxynitrite                are
other oxidants relevant to LDL oxidation                produced     by endo-
thelial cells and macrophages.              It appears that peroxynitrite                                        t/:=\!!AI                 Conlugated
increases the modification           of LDL [15]. However,         stimulated                                     S
macrophages        producing    increased nitric oxide oxidize LDL to a                                                          Uptake
lesser extent than resting cells, and inhibitors              of nitric oxide
synthase     increase LDL oxidation by activated macrophages                [16].                                V\A1                      Peroxy radical:
                                                                                                                                           abstracts H from
Thus, LDL can be oxidatively modified by numerous                     different
mechanisms.        To date, however, there is no consensus               on the                                                            anotherfatty acid
                                                                                                                                           initiating   an
predominant        mechanism      of LDL oxidation in vivo.                                                                                autocatatytic chain
     In vitro, LDL can be modified oxidatively in the presence of                                                                          reaction
transition     metals such as iron and copper. LDL oxidized by a
cell-free system is physiochemically             and biologically     indistin-                                  V=A=i
guishable from LDL oxidized by a cellular system [2]. In vitro,                                                   o                        Hydroperoxide:
LDL can bind copper, which can promote rapid lipid peroxida-                                                      I                        fragments to aldeflydes
tion [17]. LDL can be oxidized in a metal-independent                    system                                   o                        and polymenzation

with 2,2’-azobis(2-amidinopropane)              (AAPH),      a water-soluble
                                                                                                                  I                        products
                                                                                                                  H
azo compound          that thermally     decomposes,     leading to the for-
                                                                                     Fig. 1. Peroxidation of a PUFA.
mation of aqueous peroxyl radicals at a constant rate [18].
                                                                                     Adapted from ref. 22.
     The oxidizability      of LDL also depends on its size. Subjects
with a predominance          of small, dense LDL exhibit a greater risk
of coronary        artery disease compared         with individuals      with a
                                                                                     rearrangement,         leading to the formation         of conjugated  double
predominance         of large, more buoyant LDL [19]. Studies from
                                                                                     bonds referred         to as conjugated  dienes        (CD) [6]. During this
numerous   laboratories    have shown that           small,   dense    LDL     is            phase of LDL oxidation, the rate of oxidation is
                                                                                     initiation
more susceptible    to oxidation [20, 21].
                                                                                              by the presence of endogenous
                                                                                     suppressed                                 antioxidants within
                                                                                     the LDL particle, which results in the lag phase of oxidation.
                      Oxidative Modification of LW.
                                                                                     The lag phase is followed by a rapid propagation                        phase, which
Human LDL is defined as the population               of lipoproteins       that
                                                                                     occurs when         the antioxidants   are depleted    and involves the
can be isolated by ultracentrifugation        within a density range of
                                                                                     abstraction    of    another I-F by a PUFA-peroxyl        radical (LOOrn)
 1.019-1.063       kgfL [6]. Each LDL particle            contains     -1600
                                                                                     from another        PT.JFA, resulting in the formation     of lipid perox-
molecules      of cholesteryl     ester and 170 molecules       of triglycer-
                                                                                     ides. A typical      time course of copper-catalyzed     LDL oxidation,
ides, which form a central lipophilic           core. This core is sur-
                                                                                     depicting both lag and propagation      phases, including measures
rounded      by a monolayer           of -700  phospholipid       molecules,
                                                                                     of both lipid and protein oxidation,     is shown in Fig. 2. These
consisting     mainly of lecithin and small amounts of sphingomy-
elm and lysolecithin          and 600 molecules      of free cholesterol.            indices of oxidation will be discussed in detail later. Cholesterol
Embedded        in the outer layer is a large protein, apolipoprotein                in LDL can be oxidized to oxysterols such as 7-ketocholesterol
(apo) B-lOO, consisting of 4536 amino acid residues. The total                       [23]. The propagation    phase is followed by a decomposition       or
number     of fatty acids bound in different          classes of an LDL              degradation   phase, during which there is cleavage of double
molecule is -2700,         half of these being PUFAs, mainly linoleic                bonds, resulting    in the formation     of aldehydes.   The major
acid. Variations     in PIJFA content contribute       to the difference in          aldehydes produced include malondialdehyde (MDA), 4-hy-
oxidation     behavior of different LDL samples. The PI.JFAs in                      droxynonenal       (HNE), and hexanal, which can cross-link   with
LDL are protected             against free radical damage         by several         amino groups on apo B-l00.
antioxidants, the predominant one being ct-tocopherol.                                   Changes in the protein moiety also occur during the oxida-
    Oxidation of LDL is a free radical-mediated process, result-                     tion of LDL [6, 24]. After oxidation, there is an increase in the
ing in numerous structural changes, all of which depend on a                         negative     charge on the LDL particle,       possibly due to the
common initiating  event, the peroxidation of PUFAs in LDL.                          derivatization     of positively charged amino groups through the
The peroxidation  of a PUFA is shown in Fig. 1. Oxidation   of                       formation of a Schiff base with aldehydes. Also, after oxidation,
LDL is initiated by reactive oxygen species that abstract a H’                       apo B-100 undergoes              oxidative scission, leading to fragmenta-
from a double bond in PUFA, which is followed by molecular                           tion.
500                                       Jialal and Devaraj:       LDL      oxidation,   antioxidants,     and atherosclerosis




                                                                             9001         lesion. Ox-LDL     could also promote atherogenesis by altering
                                                                                    a     expression  of other genes in the arterial wall. In addition to
                                                                                    2
                                                                             750          leukocyte   adhesion molecules    M-CSF and MCP-1,     Ox-LDL
                                                                                    E     can stimulate interleukin-l    (IL-l) release from macrophages
                                                                             600
                                                                                          [26]. IL-lb    has been shown to induce smooth      muscle cell
                                                                             450 a’       proliferation      and endothelial       adhesiveness to leukocytes [27]. In
                                                                                          addition,       IL-lb   mRNA       has     been   found     in   atherosclerotic
                                                                            J300
                                                                                    0.    lesions.
                                                                                               Ox-LDL        can adversely affect the coagulation             pathway by
                                                                                          inducing    tissue factor [28] and plasminogen      activator inhibitor-i
                                                                                          synthesis    [29]; also, products of Ox-LDL       can impair expression
                                                                                          of inducible genes such as tumor necrosis factor and platelet-
                                                                                          derived growth factor [30]. Ox-LDL              inhibits     endothelium-
                                                                                          derived relaxation factor (EDRF)-mediated            vasorelaxation     [31].
                                                                                          EDRF appears to be crucial in maintaining coronary vasodila-
                                                                                          tion, and its activity is impaired in hypercholesterolemia               and
                                                                                          atherosclerosis.     Another atherogenic   property     of modified LDL
                                                                                          is its immunogenicity.      MDA-modified      LDL has been shown to
                                                                                           stimulate formation       of autoantibodies,       and immune complexes
                                                                                          of LDL aggregates are efficiently internalized                 by macrophages
                                                                                          via Fc receptors [32]. This could promote                  further cholesterol
                                                                                          accumulation.
                                                                                               Several lines of evidence support the in vivo existence of
                                                                                          Ox-LDL         [2-6]. LDL extracted          from human          atherosclerotic
                                                                                          lesions exhibits        many immunological,            physicochemical,          and
Fig. 2. Typical time course of LDL oxidation showing lag, propagation,                    biological properties         of LDL oxidized in vitro, such as cross-
and decomposition phases: (A) CD and lipid peroxide formation; (B)                        reactivity with antibodies          to MDA-lysine        conjugates,     presence
measurement of apo B fluorescence during LDL oxidation and refative                       of oxidized lipid and apo B fragments, increased electrophoretic
electrophoretic mobility (REM) of oxidized LDL.                                           mobility,     increased     uptake by macrophages,            and chemotactic
LDL (200 mg/L protein) was incubated with 5 imol/L            copper in phosphate-        activity towards monocytes            [5]. Oxidatively     modified apo B has
buffered saline for 8 h.                                                                  also been isolated from plasma of healthy subjects and patients
                                                                                          with atherosclerosis         [33]. Antibodies      against epitopes on Ox-
                       Blologkal Effects of Ox-LDL                                        LDL recognize           material from atherosclerotic           lesions but not
Ox-LDL        exerts several biological effects that may contribute                 to    from healthy arteries [34]. Ox-LDL has also been demonstrated
the initiation       and progression           of the atherosclerotic        process      in plasma of Watanabe              heritable   hyperlipidemic        rabbits and
[2-6]. A schema depicting the role of Ox-LDL in atherogenesis                             humans [34]. The presence of autoantibodies                   against Ox-LDL
is shown in Fig. 3. During                 the oxidation      of LDL, initially,          has been positively correlated            with the progression         of athero-
minimally modified LDL (MM-LDL)                        is formed in the suben-            sclerosis, as manifested         by carotid artery stenosis [35]. Also, the
dothelial space. MM-LDL                is typified by mild lipid peroxidation             oxidative susceptibility        of LDL varied with severity of coronary
and uptake by the classical LDL receptor. MM-LDL                        can induce        atherosclerosis       as evaluated by angiography          [36]. The oxidative
leukocyte-endothelial           adhesion and secretion of monocyte che-                   susceptibility      of LDL appears to be increased with established
motactic protein- 1 (MCP- 1) and macrophage                      colony-stimulat-         coronary artery disease risk factors such as diabetes, smoking,
ing factor (M-CSF)            by the endothelium           [25]. This results in          hypertension,        and hyperlipidemia      [3 7-40]. Finally, antioxidants
monocyte        binding     and recruitment          to the endothelium           and     such as probucol,             a-tocopherol,      butylated      hydroxytoluene
subsequent        migration       into the subendothelial            space, where         (BHT), and N,N’-diphenyl              phenylenediamine        have been shown
M-CSF promotes their differentiation                  into tissue macrophages.            to decrease the degree of LDL oxidation                     and atheromatous
Macrophages         in turn can modify MM-LDL                   into a more oxi-          lesions in animal models of atherosclerosis.
dized form. Ox-LDL               is no longer recognized             by the LDL
receptor; instead it is taken up by the scavenger receptor on the                                            Measurement of LDL Oxidation
monocyte-macrophages,                and this uptake is not regulated               by     The evaluation      of LDL oxidation        in vivo is fraught with
intracellular      cholesterol       content.     This results in appreciable              difficulties. One of the main problems             is that lipoprotein
cholesterol      accumulation        within the macrophages,           resulting in        oxidation is likely to occur in the mileu of the artery wall, rather
foam cell formation.           Ox-LDL         is a potent chemoattractant          for     than in the general circulation.       Even if some lipoproteins      are
monocytes        and a potent           inhibitor    of macrophage         motility,       oxidized in the circulation,   the concentrations     of these modified
thereby promoting          retention of macrophages           in the arterial wall.        lipoproteins   may be difficult to detect and may not reflect the
Ox-LDL is cytotoxic, which could promote endothelial dysfunc-                              extent of oxidation occurring       in the arterial wall. Also, exten-
tion and the evolution of the fatty streak into a more advanced                            sively modified lipoproteins     are rapidly cleared from the circu-
                                                                    Clinical Chemistry            42, No. 4, 1996                                                           501




                          4   PAIl            4     Tissue Factor
                                                                                                   CONJUGATED       DIENES
                                                                                                   One of the most widely used methods            for monitoring      LDL
                                                                                                   oxidation in vitro has been the measurement          of CD [44]. This
                                                                                                   method is rapid and easily performed.       Oxidation    of PUFA side
                                                                                                   chains of LDL is accompanied        by the formation     of dienes that
                                                                                                   absorb ultraviolet light at 234 nm. Since Ox-LDL          remains fully
                                                                                                   soluble in buffer, the increase of 234-nm diene absorption           can
                                                                                                   be measured     directly in solution,   without extraction     of LDL
                                                                                                   lipids. The typical time course of copper-mediated         LDL oxida-
                                                                                                   tion (Fig. 2) shows a lag phase, in which diene absorption        shows
                                                                                         >         only a slight increase,    followed    by a propagation       phase in which
                                                                                                   234-nm absorption     rapidly increases. In succession, the 234-nm
                                                                                     ‘             absorption  decreases, then increases again in the decomposition
                                                                                                   phase, because the aldehydes      formed also absorb in the 210-
                                                                                         =         240-nm region. Currently,   this appears to be the best index of
                                                                                                   LDL oxidizability and is clearly the most popular.

                                                                                                   SPECTROPHOTOMETRIC             ASSAY    FOR    LIPID     PEROXIDES
                                                                                                   This rapid and simple method is based on the oxidation of iodide
                                                                                                   to iodine by lipid peroxides formed during oxidation of LDL
                                                                                                   [45]. However, the disadvantage  is that the lower detection limit
                                Foam Cell                                                          is similar  to the reagent blank and the method         measures other
                                                                                                   peroxides as well.
    Smooth Muscle Cell                                                                                 Another iodometric      assay deals with the problem of specific-
                                                                                                   ity by hydrolyzing     esterified   lipids and extracting     them with
Fig. 3.   Schema depicting    role of Ox-LDL in atherogenesis.
                                                                                                   ethyl acetate before determination        of their hydroperoxide   con-
                                                                                                   tent [46]. The ferrous ion oxidation assay [47], in which ferrous
lation by scavenger receptors, and therefore their residency in                                    ions are oxidized in the presence of xylenol orange, also seems to
the plasma may be too short-lived   and the concentrations   too                                   be relatively specific for lipid peroxides. The most specific and
low for easy measurement.            In experimental       animals,        samples           of    sensitive assay for determination    of lipid peroxides in biological
                                                                                                   fluids is HPLC with isoluminol        chemiluminescence      detection
arterial  tissue can be obtained             to examine           the amount     of
                                                                                                   [48]. However,    this assay is time consuming      and is not readily
oxidative    modification; however,          in humans,           limited samples
                                                                                                   adaptable    to the clinical   laboratory     setting.
(blood,    urine,   and   expired     air) are available.          In view of the
increasing interest in the role of LDL oxidation in the patho-
                                                                                                   THIOBARBITURIC        ACID-REACTIVE           SUBSTANCES       (TBARS)
genesis of atherosclerosis, there is clearly a need for improved
                                                                                                   ASSAY
methods      to evaluate lipoprotein      oxidation,         especially in vivo,
                                                                                                   The most commonly     used assay in LDL oxidation studies, both
particularly    at extravascular    sites. Because           of the difficulties
                                                                                                   in the presence and absence of cells, is the TBARS test [49]. In
encountered   in obtaining    tissue samples              in humans,      several
                                                                                                   this  test, the chromogen  is formed   by the reaction  of one
indirect measures of lipoprotein     oxidation            and antioxidant     po-
                                                                                                   molecule    of MDA with two molecules    of TBA. The method
tential in vivo must be used and are discussed                    below.    Recently,              involves heating the sample with TBA under acidic conditions
we described    various measures of quantifying                    LDL      oxidation
                                                                                                   and reading the absorbance  of the MDA-TBA    adduct formed at
                                                                                                   532 nm. This test is not specific for MDA, since sugars and
     LDL is isolated from plasma in EDTA (I g/L) either by                                         amino acids may also form TBA adducts; furthermore,            a signif-
sequential ultracentrifugation     in NaBr solutions [42] or by rapid                              icant amount of peroxides are formed during the heating step of
vertical spin gradient ultracentrifugation   [43]. The isolated LDL                                the assay. Various modifications         of the TBA test have been
is extensively   dialyzed agianst NaCI-EDTA,        pH 7.4, filtered,                              proposed,    involving differences    in sample treatment,   acid con-
and stored               after
                at 4 #{176}C purging         with     nitrogen.      To eliminate                  centration,    heating time, and presence or absence of antioxi-
EDTA      before the oxidation   experiments,   LDL is dialyzed                                    dants.
overnight    against two changes   of phosphate-buffered  saline                                        The spectrophotometric      test of TBARS [50] includes precip-
                                        or
(PBS) at pH 7.4, in the dark at 4 #{176}C passed through    a                                      itation of protein with trichioroacetic       acid. The assay is con-
Sephadex G-2 5 column (Pharmacia, Piscataway,  NJ). For oxi-                                       ducted in the presence of BHT-EDTA              to minimize peroxide
dation of LDL by copper, filtered LDL (200 mgfL) is incubated                                      formation    due to heating.
          in
at 37 #{176}CPBS for 8 h in a time-course   experiment,  with 5                                         To increase sensitivity,   the MDA-TBA         adduct can be ex-
.tmol/L copper. Oxidation is stopped at the various time points                                    tracted into an organic solvent (butanol) and measured fluoro-
by BHT-EDTA,     followed by refrigeration.                                                        metrically [51]. Although this test is widely used to assess lipid
502                                     Jialal and Devaraj:   LDL     oxidation,   antioxidants,   and atherosclerosis




peroxidation, it lacks specificity         and should    not be the only           This is a good measure of LDL                oxidation    but is not easily
measure used.                                                                      adaptable to the routine laboratory.

RELATIVE         ELECTROPHORETIC       MOBILITY                                    OXYSTEROLS
LDL has a negatively charged surface and migrates to the anode                     Oxidative modification    of LDL can also be assessed by measur-
in agarose gel electrophoresis        under nondenaturing      conditions.         ing oxidation products of cholesterol,     oxysterols [56]. Although
Oxidation       renders    LDL more negatively        charged,    possibly         several are produced,    7-ketocholesterol    has been identified    as
because of derivatization      of lysine residues of apo B- 100 by some            the main oxysterol produced during copper-catalyzed          and cell-
reactive aldehydes formed during oxidation, and accordingly                its     mediated    oxidation  of LDL and can be measured          by GC or
electrophoretic       mobility increases (Fig. 2). Another possibility             GC-mass    spectrometry (MS) methods.  Measurement   of oxy-
for the increase of negative charge on LDL during oxidation is                     sterols in human plama can become a test of LDL oxidation.
that reactive oxygen species generated            convert histidine     and        However, not much data have been reported in this regard.
proline residues to negatively charged aspartate or glutamate. A
measurable index of this is the relative electrophoretic           mobility,       F2 -ISOPROSTANES
which is the ratio of migration    distance of oxidized            to native       It was recently discovered        that a series of structurally      unique
LDL.      This    is a very reliable  way to quantify LDL oxidation in             prostaglandin   F2 (PGF2)-like       compounds     (F2-isoprostanes)      are
vitro but it clearly lacks the sensitivity       of an in vivo test. Other         produced in vivo in humans by a noncyclooxygenase               mechanism
aldehydic modifications          will also alter the electrophoretic   mo-         involving free radical-catalyzed      peroxidation   of arachidonic acid.
bility of LDL.                                                                     Of these, 8-epi-PGF2-a,          the major component,          is a potent
                                                                                   vasoconstrictor  [57]. The release of PGF2 is increased in LDL
APO     B-ba      FLUORESCENCE
                                                                                   oxidized by macrophages,      endothelial   cells, or copper and can be
The oxidative modification       of LDL also generates fluorophores,
                                                                                   measured     by a solid-phase    extraction    procedure,      followed by
which fluoresce strongly at 430 nm with excitation at 360 nm,
                                                                                   GC-MS      [57].The   formation    of F2-isoprostanes       is induced in
owing to derivatization     of apo B- 100 lysine residues by reactive
                                                                                   plasma and LDL exposed to oxidative stress in vitro [58]. Also,
aldehydes [52]. A typical time course of apo B fluorescence           is
                                                                                   as was recently shown, F2-isoprostanes        and their metabolites     are
shown in Fig. 2. This assay has the same problems as discussed
                                                                                   increased    in plasma and urine of smokers               [59]. However,
for measuring    electrophoretic     mobility. Although   it is a very
                                                                                   although this is a measure of LDL oxidation, measurement                  in
reliable index of protein modification       of LDL during oxidation,
                                                                                   urine reflects whole-body     oxidation rather than LDL oxidation.
the assay is not sensitive enough to measure basal LDL oxida-
                                                                                   Since this novel method offers a lot of promise, research should
tion.
                                                                                   be directed at developing   a plasma assay that can be adapted to
                                                                                   the clinical laboratory.
FAYY       ACID    CONTENT
                                                                                       Specific fluorescence patterns can be produced when certain
Since oxidative modification       of LDL is essentially a free radical-
                                                                                   amino acids react with lipid peroxides.     Previously, dityrosine
mediated process involving oxidation of PI.JFAsin LDL, mea-
                                                                                   fluorescence    was shown to be associated      with oxidation     of
surement of the fatty acid content could be an indication of the
oxidative susceptibility     of the LDL particle. About half of the                linoleic acid [60]. Phagocytes generate myeloperoxidase               to kill
                                                                                   invading bacteria; this may convert tyrosine to a radical            catalyst
fatty acids in LDL are PI.JFAs, mainly linoleic acid and minor
amounts     of arachidonic      and docosahexaenoic        acids. Dietary          that cross-links    proteins.  The stable oxidized product of the
habits confer a large degree of interindividual         variability in the         tyrosyl radical is dityrosine; its stability and intense fluorescence
LDL fatty acid composition.         Therefore,   it is useful to monitor           may allow it to also act as a marker for oxidatively          damaged
the disappearance     of these three main fatty acids. Fatty acids are             proteins in lesions. This method could prove useful in evaluating
measured      by gas chromatography         (GC) after extraction      and         the role of specific protein      modifications    that occur during
transmethylation     [53]. However,       this instrumentation      is not         lipoprotein   oxidation.
generally available in clinical laboratories     and the method is very
time consuming.                                                                                               Antioxidant Status
                                                                                   ANTIOXIDANTS        AND    LDL   OXIDATION

ALDEHYDES                                                                          The antioxidant     content of LDL is critical for its protection. In
It has been proposed          that aldehydes such as MDA or FINE,                  theory, if sufficient lipophilic antioxidants   were present, LDL
generated    by lipid peroxidation       from Pl.JFAs in LDL, interact             would be protected from even profound oxidant challenge. The
with apo B and specifically           modify lysine residues [54]. For             balance between the prooxidant        challenge and the presence of
measurement       of aldehydic lipid peroxidation        products, LDL is          antioxidants  determines    the extent of arterial wall modification
derivatized    with dinitrophenyl       hydrazine,    and the hydrazones           of LDL. Antioxidants      such as probucol,     N,N’-diphenyl     phen-
are extracted      with dichloromethane,         separated    by thin-layer        ylenediamine,   and BHT have been shown to decrease the degree
chromatography,        and analyzed by HPLC with an ODS column                     of oxidation and the extent of atheromatous          lesions in animal
(e.g., Ultrasphere      absorbance    Spherisorb    column; Waters, Mil-           models of atherosclerosis      [61], but have side effects. Thus,
ford, MA) and eluted with acetonitrile:          water (9:10, byvol). The          dietary antioxidants     such as a-tocopherol,        a-carotene,   and
effluent is monitored       at 223 nm and HNE can be identified [55].              ascorbic    acid become    attractive   alternatives.    We have reviewed
                                                                   Clinical Chemistry         42, No.    4, 1996                                                           503




extensively  in previous          reports        the   role   of antioxidants            in    13-Carotene. 13-Carotene,  a hydrophobic   member of the carote-
relation to atherosclerosis        [5].                                                        noid family, is carried in the blood, mainly in LDL. Esterbauer
                                                                                               et al. [62] have shown that carotenoids  provide auxiliary antiox-
cr-Tocopherol. a-Tocopherol     (vitamin E) is the principal lipid-                            idant defenses with respect to LDL after cr-tocopherol.     Jialal et
soluble antioxidant   in plasma and in the LDL particle [62]. It is                            al. [82] have shown that properly      dissolved  13-carotene     can
a chain-breaking    antioxidant    and traps peroxyl free radicals.                            inhibit LDL oxidation in vitro induced by copper or by macro-
Several studies have associated low a-tocopherol       concentrations                          phages. Preincubation    of cocultures  of endothelial    cells and
with the development     of atherosclerosis. A cross-sectional     study                       smooth muscle cells with 13-carotene prevented    LDL modifica-
of  16 European  populations    has shown a significant                   correlation          tion and its induction    of monocyte    transmigration [83]. The
between a-tocopherol     concentrations  and mortality                    from coro-           supplementation   studies with 13-carotene have been disappoint-
nary artery disease [63]. A previous study has shown an inverse                                ing with respect to the protection    of LDL from oxidation,     in
correlation   between plasma vitamin E concentrations        and the                           contrast to the studies with a-tocopherol.
risk of angina pectoris [64]. Vitamin E supplementation     has been
shown to reduce the risk of coronary artery disease in men and                                 Other antioxidants. Flavonoids are plant-derived     compounds    that
women [65, 66], but not in middle-aged      smokers from Finland                               inhibit in vitro copper-catalyzed,   ultraviolet-induced,      macro-
who were receiving 50 mg/day of a-tocopherol        for 5 years. The                           phage-mediated    LDL oxidation      [84, 85]. The inhibition     of
failure of the Finnish study may be attributed   to the long period                            oxidation of human LDL by consumption         of red wine or tea has
during which the study population   was at risk, the relatively late                           been attributed  to the presence    of antioxidants     such as fla-
time point when supplementation      was initiated,    and probably                            vonoids and other polyphenols    in red wine and catechin in tea.
too low a dose of vitamin      E administered       to inhibit LDL                             Ubiquinol-lO    is another effective lipid-soluble  antioxidant     that
                                                                                               inhibits LDL oxidation due to aqueous or lipid-phase           peroxyl
oxidation.  Some animal studies              [67, 68] have also shown that
                                                                                               radicals [86]. However, further studies, especially in humans, are
dietary a-tocopherol  can retard             the progression  of atheroscle-
                                                                                               required to validate the role of these antioxidants     in inhibiting
rosis.
                                                                                               LDL      oxidation.
     a-Tocopherol  has been shown to inhibit LDL oxidation in
vitro, and supplementation   of human volunteers     with a-toco-
                                                                                               MEASUREMENT           OF ANTIOXIDANTS
pherol has been shown to decrease the susceptibility      of their
                                                                                               A good      measure    of the antioxidant       capacity     of LDL     can be
LDL to oxidation [5]. A recent dose-response    study shows that
                                                                                               derived from its antioxidant    content. The lag phase of oxidation
at least 400 lU/day     is required  to significantly    decrease the
                                                                                               is directly proportional  to the antioxidant    content of LDL and is
susceptibility   of LDL to oxidation [69].
                                                                                               related by the equation y = a + kx, where y                lag time in
    a-Tocopherol     may also have additional    benefits for cardio-
                                                                                               minutes,    a = other antioxidants        present    in LDL such as
vascular disease. a-Tocopherol,     alone or in combination       with
                                                                                               13-carotene or ubiquinol,          k = efficiency        constant  of a-toco-
ascorbate  and 13-carotene,  has been shown to reduce platelet
                                                                                               pherol, and x = amount of a-tocopherol                (mol/mol)   in the LDL
adhesion [70]. Physiological concentrations      of a-tocopherol also
                                                                                               [6]. Measurement       of a-tocopherol,       retinol, and five carotenoids
inhibit smooth muscle proliferation,      protein kinase C activity
                                                                                               (lutein, cryptoxanthin,       lycopene, and a- and 13-carotene) can be
[71], and agonist-induced         monocyte        adhesion     to cultured      human
                                                                                               performed      by reversed-phase       HPLC. Simultaneous          determina-
endothelial  cells [72J. Low-dose     supplementation     with a-toco-                         tion of these antioxidants        is possible after ethanol precipitation
pherol has been shown to preserve            endothelium-dependent
                                                                                               and hexane extraction          of plasma or LDL. The hexane phase
vasodilation  in hypercholesterolemic      rabbits [73].                                       is evaporated,      reconstituted      in ethanol,      passed on a HPLC
                                                                                               reversed-phase       C18 column,          and eluted        with acetonitrile:
Ascorbate.     Ascorbate    (vitamin        C)     is a water-soluble,          chain-         dichloromethane:methanol             (70:20:10     by vol) [87]. Retinol       is
breaking     antioxidant    that regenerates      a-tocopherol     from its                    measured at 325 nm, a-tocopherol               at 292 nm, and the carote-
chromanoxyl       radical form [74]. Low plasma and tissue concen-                             noids at 450 nm.
trations of ascorbate       have been identified       as a risk factor for                         Another index of the antioxidant          status of plasma is the total
atherosclerosis.     Plasma ascorbate has been shown to be inversely                           radical-trapping      antioxidant      parameter      [88]. This is a total
correlated     with coronary      disease mortality     [61]. Further,  the                    estimate of antioxidants         present in plasma, such as ascorbate,
concentrations       of ascorbate    in atheromatous      aortas are lower                     urate, sulfhydryls, and a-tocopherol.           The activity of antioxidant
than in control vessels [75]. Smokers, diabetics, and patients with                            enzymes such as superoxide dismutase, catalase, and glutathione
coronary artery disease all have lower concentrations     of plasma                            peroxidase is also determined.          However, this assay does not give
ascorbate [76-78].                                                                             information      on the individual antioxidants.
    Physiological  concentrations            of ascorbate       can inhibit       LDL
oxidation by copper or cultured             macrophages         [79], and can also                                   Other Measures of Oxidation
inhibit oxidation  by activated             neutrophils        or U937 cells or                Another potential  way to evaluate lipoprotein     oxidation  is by
AAPH [80]; both systems lack metal catalysts.                   Dietary      ascorbate         measurement    of autoantibodies  against epitopes     on oxidized
supplementation        has been      shown        to prevent      LDL        oxidation         LDL [89] by ELISA. A recent study has shown that the titer of
induced      by acute cigarette     smoking        [81].                                       these antibodies      is an independent      predictor     of the progression
504                                       J ialal and Devaraj: LDL oxidation, antioxidants,             and atherosclerosis




of carotid atherosclerosis  in patients with accelerated atheroscle-                      lipase A2 mimicks cell-mediated   oxidative modification.   J Lipid
rosis [90].                                                                               Res 1988;29:749-53.
     Nuclear magnetic resonance analysis of oxidized lipoproteins                     12. VIa Herttuala 5, Rosenfeld ME, Parthasarathy   S, Glass CK, Sigal
                                                                                          E, Witztum JL, Steinberg 0. Colocalization of 15-lipoxygenase
[91] could provide interesting     details concerning   the structural
                                                                                          mRNAand protein with epitopes of oxidized low density lipoprotein
aspects of these modified lipoproteins.      Breakdown     products of                    in macrophage-rich areas of atherosclerotic lesions. Proc Natl
lipid peroxides are exhaled in breath as volatile hydrocarbons.                           Acad Sd U S A 1989;86:1046-50.
The volatile oxidation      products of n-6 and n-3 fatty acids,                      13. Savenkova Ml, Mueller DM, Heinecke JW. Tyrosyl radical gener-
pentane and ethane, appear in breath and can be measured by                               ated by myeloperoxidase: a physiological catalyst for the initiation
GC [92]. This method is sensitive and noninvasive;      however, it                       of lipid peroxidation in low density lipoprotein. i Biol Chem
                                                                                          1994;269:2O394-400.
is tedious and one must purify the inspired air because of the
                                                                                      14. Hazfil U, Stocker R. Oxidation of low density lipoprotein with
high background      created by exogenous   sources such as motor
                                                                                            hypochlorite causes transformation of the lipoprotein into a high
vehicles and cigarette smoke. Also, it is a measure of whole-body                         uptake form for macrophages.        Biochem J 1993;90:165-72.
oxidation and not LDL oxidation specifically.                                         15. Hogg N, Darley-Usmar DM, Graham A, Moncada S. Peroxynitrite
                                                                                          and atherosclerosis.    Biochem Soc Trans 1993;21:358-62.
In conclusion,    it has become increasingly        evident that oxidation            16. Jessup W, Mohr D, Gieseg SP, Dean RT, Stocker R. The partici-
of LDL is a key step in atherogenesis.                Although      there are             pation of nitric oxide in cell-free and its restriction of macrophage-
                                                                                          mediated oxidation of LDL by mouse macrophages.              FEBS Lett
various ways to measure oxidative modification                of LDL, each
                                                                                          1992;309:135-9.
method has its limitations. The easiest measure of oxidation that
                                                                                      17. Steinbrecher     UP, Witztum JL, Parthasarathy         5, Steinberg D.
can be adapted to the clinical laboratory is the measurement                of            Decrease in reactive amino groups during oxidation or endothelial
CD by continuous        absorption    spectrophotometry.         This can be              cell modification    of LDL: correlation with changes in receptor
monitored     during copper-catalyzed        oxidation of isolated LDL.                     mediated catabolism. Arteriosclerosis 1987;7:135-43.
The measurement         of isoprostanes     and ethane and methane in                 18. Frei B, Stocker R, Ames BN. Antioxidant defenses and lipid
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                                                                                          1988;85:9748-54.
direct test so far that documents       oxidation, e.g., LDL oxidation,
                                                                                      19. Austin MA, Breslow JL, Hennekens CH, Buring JE, Willett WC,
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                                                                                          Krauss RM. LDL subclass patterns and risk of myocardial infarc-
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measure     of LDL oxidation         that could become part of the                    20. Tribble DL, Holl LG, Wood PD, Krauss RM. Variations in oxidative
laboratory       repertoire  in the diagnosis      and management       of                susceptibility of LDL subfractions of differing density and particle
atherosclerosis.                                                                          size. Atherosclerosis 1992;93:i89-99.
                                                                                      21. Chait A, Brazg RL, Tribble DL, Krauss RM. Susceptibility of small,
                                                                                          dense low density lipoproteins    to oxidative modification in sub-
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