© 1995 American Heart Association, Inc.
Articles |
Inhibition of Lecithin-Cholesterol Acyltransferase and Modification of HDL Apolipoproteins by Aldehydes
From the Department of Molecular and Nuclear Medicine, Life Sciences Division, Lawrence Berkeley Laboratory, University of California at Berkeley.
Correspondence to Mark R. McCall, PhD, Lawrence Berkeley Laboratory, Donner Laboratory, 1 Cyclotron Rd, Berkeley, CA 94720.
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Abstract |
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Abstract Experimental evidence suggests that aldehydes generated as a consequence of lipid peroxidation may be involved in the pathogenesis of atherosclerosis. It is well documented that aldehydes modify LDL; however, less is known concerning the effects of aldehydes on other plasma and interstitial fluid components. In the present study, we investigated the effects of five physiologically relevant aldehydes (acetaldehyde, acrolein, hexanal, 4-hydroxynonenal [HNE], and malondialdehyde [MDA]) on two key constituents of the antiatherogenic reverse cholesterol transport pathway, lecithin-cholesterol acyltransferase (LCAT) and HDL. Human plasma was incubated for 3 hours at 37°C with each one of the five aldehydes at concentrations ranging from 0.16 to 84 mmol/L. Dose-dependent decreases in LCAT activity were observed. The short-chain (acrolein) and long-chain (HNE)
,ß-unsaturated aldehydes were the most effective LCAT
inhibitors. Micromolar concentrations of these unsaturated
aldehydes resulted in significant reductions in plasma LCAT activity.
The short- and longer-chain saturated aldehydes acetaldehyde and
hexanal and the dialdehyde MDA were considerably less effective at
inhibiting LCAT than were acrolein and HNE. In addition to inhibiting
LCAT, aldehydes increased HDL electrophoretic mobility and
cross-linked HDL apolipoproteins. Cross-linking of
apolipoproteins A-I and A-II required higher aldehyde concentrations
than inhibition of LCAT. The ,ß-unsaturated aldehydes acrolein
and HNE were fourfold to eightfold more effective cross-linkers of
apolipoproteins A-I and A-II than the other aldehydes studied. These
data suggest that products of lipid peroxidation, especially
unsaturated aldehydes, may interfere with normal HDL
cholesterol transport by inhibiting LCAT and modifying HDL
apolipoproteins.
Key Words: lecithin-cholesterol acyltransferase • reverse cholesterol transport • apolipoproteins A-I and A-II • ,ß-unsaturated aldehydes • HDL
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Introduction |
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Plasma HDL concentrations are inversely correlated with coronary heart disease risk.1 2 The mechanism for a direct protective effect of HDL has not been firmly established, although considerable evidence links its protective effects to the antiatherosclerotic reverse cholesterol transport pathway (for recent reviews see References 3 through 63 4 5 6 ). HDL has multiple functions in this putative pathway: it facilitates the efflux and net transfer of excess cholesterol from atherosclerotic foam cells; it provides the activator (apo A-I) and substrates (ie, cholesterol and phospholipid) for LCAT, the enzyme thought to be responsible for establishing the unesterified cholesterol concentration gradient along which foam cell–derived cholesterol flows; and it facilitates the transport of foam cell–derived cholesterol to the liver for reutilization or catabolism.
Recent in vivo evidence implicates lipoprotein oxidation in the pathogenesis of atherosclerosis. Oxidized forms of LDL have been detected in atherosclerotic lesions from both animals7 8 9 10 11 and humans.11 Moreover, lipophilic antioxidants have been shown to retard the development of atherosclerosis in hypercholesterolemic animals.12 13 14 15 In vitro studies tend to support these in vivo observations, since it has been demonstrated that oxidized LDLs are chemotactic for monocytes16 17 18 19 and also promote the ex vivo conversion of monocytes into lipid-laden foam cells.20 21 The molecular mechanisms responsible for the increased atherogenicity of oxidized LDLs have not been fully explored, although products of LDL lipid peroxidation are almost certainly involved. A variety of reactive aldehydic products (eg, HNE, MDA) are generated as a consequence of LDL oxidation.22 These products resulting from the oxidative degradation of LDL polyunsaturated fatty acids have been shown to be capable of covalently attaching to the lysine residues of apo B,23 24 25 26 the major structural protein of LDL. Such modified LDLs have increased agarose anodic mobility and tend to be taken up in an unregulated manner by macrophages,23 24 26 giving rise to foam cells.
Information concerning the effects of oxidation on HDL is limited. Recent studies, however, have shown that HDL is not only more easily oxidized than LDL27 but may also accept peroxidized lipids from LDL incubated with endothelial cells.28 These reports provide suggestive evidence that HDL, in addition to LDL, may be affected by oxidative events in vivo. One possible consequence of transport of lipid hydroperoxides by HDL is that HDL may be exposed to the aldehydic decomposition products of these oxidized lipids. Although aldehyde-modified HDLs are not taken up to any great extent by the scavenger receptor of macrophages,28 it appears that aldehyde-modified HDLs are impaired in their ability both to interact with LCAT29 and to promote cholesterol efflux from cholesterol-laden cells.30 31
In the present study, we investigated whether physiologically relevant long-chain lipophilic aldehydes such as hexanal and HNE and shorter-chain hydrophilic aldehydes such MDA, acetaldehyde, and acrolein affect either plasma LCAT activity or HDL structure. Our data show that these aldehydes are indeed effective inhibitors of LCAT and cross-linkers of HDL apolipoproteins.
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Methods |
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Materials
Acetaldehyde, acrolein, aminoguanidine bicarbonate salt, ß-mercaptoethanol, BHT, cholesterol, GSSG, DTNB, EDTA, human serum albumin, and tris(hydroxymethyl)aminomethane (Tris base) were obtained from Sigma Chemical Co. MDA was obtained by acid hydrolysis of 1,1,3,3,-tetraethoxypropane (Sigma) as described by Haberland and coworkers.24 Hexanal was purchased from Aldrich Chemical Co; GSH was obtained from Gibco BRL. L-
-Phosphatidylcholine (egg) was purchased from Avanti
Polar-Lipids, Inc, and [4-14C]cholesterol was
obtained from NEN Products. HNE was a generous gift of Dr Larry
Sayre (Case Western Reserve University, Cleveland, Ohio).
Subjects
Blood was obtained with informed consent from fasted, healthy,
adult volunteers. EDTA (4 mmol/L) was used to prevent coagulation, and
plasma was separated from cellular blood components by low-speed
centrifugation (2000g, 4°C). Plasmas from
four to six volunteers were pooled, and gentamicin sulfate (50 mg/L)
was added to prevent microbial contamination. Pooled plasma was used
for all experiments; plasma concentrations for total
cholesterol, HDL cholesterol, and
triacylglycerol expressed as the mean±SD for all
four pools were 5.59±0.49, 1.29±0.08, and 1.11±0.23 mmol/L (216±19,
50±3, and 97±20 mg/dL), respectively. In some experiments, either
GSH, GSSG, or aminoguanidine was added to plasma before aldehyde
exposure; details are provided in the figure legends.
Exposure of Plasma to Aldehydes
Plasma was exposed to various concentrations of aldehydes for 3
hours at 37°C. Three hours was chosen for incubations because
preliminary experiments indicated that maximum HDL apolipoprotein
cross-linking occurred within this time interval. The
short-chain hydrophilic aldehydes acetaldehyde, acrolein, and MDA
(Fig 1
) were added to plasma in PBS. The lipophilic
longer-chain aldehydes HNE and hexanal (Fig 1) were dissolved in
chloroform immediately before use. The desired amount of aldehyde in
chloroform was subsequently transferred to a glass vial and the
chloroform evaporated under a gentle stream of nitrogen. Plasma was
immediately added to aldehyde with mixing.
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In some experiments, plasma was pretreated with the reversible LCAT inhibitor DTNB before aldehyde exposure.32 Plasma was incubated at 37°C for 0.5 hour in the presence or absence of 1.7 mmol/L DTNB; excess DTNB was subsequently removed by dialysis in PBS. Plasma LCAT activity was completely inhibited after this procedure and could be restored by addition of 5 mmol/L ß-mercaptoethanol. Control and DTNB-treated plasmas were incubated with acrolein or acetaldehyde as described above.
LCAT Activity Measurements
Plasma LCAT activity was assessed by the exogenous "common
substrate" (ie, proteoliposome) method of Albers et
al.33 This assay is dependent on the amount of active
enzyme and independent of endogenous plasma substrates and
cofactors.34 The
[14C]cholesterol-labeled proteoliposomes
used to measure LCAT activity were prepared from egg yolk
phosphatidylcholine, unesterified cholesterol, and human
apo A-I by the cholate dialysis procedure. LCAT activity was determined
by measurement of the conversion of radiolabeled
cholesterol to cholesteryl ester after incubation of a
small aliquot of plasma (7.5 µL) with 242.5 µL of a mixture
containing the labeled proteoliposome substrate. The final assay
mixture contained 4 nmol cholesterol, 125 nmol egg yolk
phosphatidylcholine, 0.4 nmol apo A-I, and 1.3 mg human serum
albumin in the following buffer (mmol/L): Tris 20 (pH 8.0),
NaCl 150, EDTA 0.27, and ß-mercaptoethanol 5. After a 30-minute
incubation at 37°C, esterified cholesterol was separated
from unesterified cholesterol by thin-layer
chromatography; radioactivity associated with these
lipids was quantified by liquid scintillation counting.
Characterization of Modified HDL
HDLs were isolated from control and aldehyde-exposed plasma
by sequential preparative ultracentrifugation as
previously described.35 Isolated HDLs were subsequently
dialyzed against a lower-salt background (150 mmol/L NaCl, 1 mmol/L
EDTA, pH 7.4) and assayed for protein.36 HDL samples were
subjected to agarose gel electrophoresis to determine whether their
electrophoretic mobility was altered by aldehyde exposure. Beckman
Paragon Lipo gels were used according to the manufacturer's
recommendations. HDL apolipoprotein cross-linking was assessed by
SDS-PAGE performed under nonreducing conditions as described by
Laemmli37 with Schleicher and Schuell 4% to 20% gels.
Western blot analyses of SDS-PAGE gels were carried out with
polyclonal antibodies monospecific for apo A-I and A-II as previously
described.35
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Results |
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Effects of Aldehydes on LCAT Activity
Exposure of plasma to increasing concentrations of either acetaldehyde, acrolein, hexanal, HNE, or MDA resulted in dose-dependent decreases in LCAT activity (Fig 2A
and 2B). The short- and long-chain ,ß-unsaturated
aldehydes acrolein and HNE are the most effective LCAT
inhibitors. Micromolar concentrations of these
unsaturated aldehydes result in significant reductions in plasma
LCAT activity (Fig 2A). The short- and longer-chain saturated
aldehydes acetaldehyde and hexanal and the dialdehyde MDA are
considerably less effective at inhibiting LCAT than acrolein and HNE.
Reductions in LCAT activity to 
10% of control require
concentrations of acetaldehyde, hexanal, and MDA >40 mmol/L.
Concentrations of acrolein and HNE required to achieve similar levels
of inhibition (ie, 10% of control) are 
10 mmol/L. Interestingly, the
dialdehyde MDA is little more effective than hexanal in promoting LCAT
inhibition. Although the effective carbonyl concentration in
incubations containing MDA is twice that of the other aldehydes
studied, it is likely that the short chain length of MDA limits the
participation of both carbonyls in adduct formation.
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Effects of Aldehydes on HDL Structure
In addition to inhibiting LCAT, aldehydes added to plasma increase
the agarose electrophoretic mobility of HDL; moreover, at the highest
aldehyde concentrations, the HDL band became more diffuse. Fig 3 shows representative agarose gels of
HDL isolated after exposure of plasma to acetaldehyde, acrolein,
hexanal, and HNE. For a given aldehyde concentration, acrolein and HNE
are more effective than acetaldehyde and hexanal at increasing anodic
mobility (note concentration differences between saturated and
unsaturated aldehydes in Fig 3). In general, the unsaturated aldehydes
that are most effective at inhibiting LCAT (ie, acrolein and HNE) are
also most effective at modifying HDL charge.
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The major structural proteins of HDL, apo A-I and apo A-II, are
cross-linked as a result of the exposure of plasma to aldehydes.
The upper panels of Fig 4 show
representative apo A-I Western blots of HDL isolated
from plasma exposed to increasing concentrations of either the
saturated aldehyde acetaldehyde or the unsaturated aldehyde acrolein.
The aldehyde concentrations required to induce cross-linking of apo
A-I (the most abundant apolipoprotein associated with HDL) differ
considerably between these two aldehydes. Acrolein induced
cross-linking of apo A-I at low concentrations, 0.625 mmol/L,
whereas acetaldehyde required eightfold greater concentrations to
induce cross-linking. The lower panels of Fig 4 show
representative SDS-PAGE gels and Western blots for apo
A-I and apo A-II of HDL isolated from plasma exposed to either 21.0
mmol/L acetaldehyde or 5.25 mmol/L acrolein. Aldehyde concentrations
were selected so that cross-linked apolipoproteins could be clearly
identified on SDS-PAGE gels. The same molecular weight forms of
cross-linked apo A-I are observed for both aldehydes. In addition,
it is also evident that both aldehydes induce cross-linking of the
second most abundant apolipoprotein associated with HDL, apo A-II. The
longer-chain-length saturated and unsaturated aldehydes hexanal
and HNE also cross-linked apo A-I and A-II (data not shown). HNE,
like acrolein, was very effective in this regard, whereas hexanal was
somewhat less effective than acetaldehyde in promoting
cross-linking. These data are consistent with our
observation that acrolein and HNE are the most effective aldehydes at
increasing HDL anodic mobility; furthermore, they suggest that the
,ß-unsaturated aldehydes are better able to form adducts with
HDL apolipoproteins than are acetaldehyde and hexanal.
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Effects of Aminoguanidine and GSH on Aldehyde-Induced LCAT
Inhibition and HDL Modification
Aldehyde adducts increase the net negative surface charge of HDL
by modifying positively charged amino acid residues (eg, lysines)
and/or by altering protein conformation, exposing negatively charged
residues or burying positively charged residues. To investigate the
role of interactions between aldehydes and protein primary amines, such
as the 
-amino group of lysine, in LCAT inhibition and HDL
cross-linking, we included the hydrazine compound aminoguanidine in
plasma incubations with acetaldehyde, acrolein, hexanal, and HNE.
Aminoguanidine has been shown to inhibit apo B lysine modification
resulting from endothelial cell– or copper-induced
LDL oxidation.38 Since LCAT was more sensitive to
inactivation and HDL was more sensitive to cross-linking by
,ß-unsaturated aldehydes than saturated aldehydes, we used
lower concentrations of acrolein and HNE than of acetaldehyde and
hexanal in these experiments. When either acrolein or HNE incubations
were supplemented with equimolar concentrations (5.25 mmol/L) of
aminoguanidine, LCAT was not protected (Fig 5).
Moreover, cross-linking of apolipoproteins on HDL was not prevented
(Fig 6; lanes 4 and 12). In contrast, when plasma
incubations with either acetaldehyde or hexanal were supplemented with
equimolar concentrations of aminoguanidine (21.0 mmol/L), both LCAT
inhibition (Fig 5) and HDL-apolipoprotein cross-linking (Fig 6;
lane 8, hexanal data not shown) were reduced. These data indicate that
saturated aldehydes such as acetaldehyde and hexanal are more
effectively scavenged by aminoguanidine than are
,ß-unsaturated aldehydes such as acrolein and HNE.
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Aldehydes are also known to form adducts with protein
thiols.39 40 We previously demonstrated that glutathione
protects LCAT activity and HDL structure from gas-phase cigarette
smoke; thus, we investigated the ability of GSH to protect LCAT and HDL
from aldehyde-induced modification. In the case of
,ß-unsaturated aldehydes, in which low concentrations inhibit
LCAT activity and modify HDL structure, we found that supplementing
plasma with 5.25 mmol/L GSH before exposure to an equivalent
concentration of HNE or acrolein resulted in substantial protection of
LCAT activity (Fig 5
) and a reduction in HDL cross-linking (Fig 6;
lanes 2 and 10). Furthermore, Fig 5 demonstrates that GSH more
effectively quenched acrolein than HNE. In the case of the saturated
aldehydes, which require higher concentrations to inhibit LCAT and
modify HDL structure, we found that supplementing plasma with 21.0
mmol/L GSH before exposure to an equivalent concentration of
acetaldehyde or hexanal resulted in protection of LCAT activity and a
reduction in apolipoprotein cross-linking (Figs 5 and 6;
cross-linking data are shown for acetaldehyde only, Fig 6, lane 6).
In contrast, the oxidized form of glutathione was unable to prevent
either LCAT inhibition or HDL-apolipoprotein cross-linking by any
of the aldehydes examined (Figs 5 and 6). This suggests that the thiol
group rather than the amino group of the GSH tripeptide participates in
adduct formation with these aldehydes. Taken together, these data
emphasize the reactivity of aldehydes toward thiols and suggest that
free cysteine residues in proteins are likely targets for aldehyde
adduct formation.
In addition, it is clear from Fig 5 that the longer-chain
lipophilic aldehydes hexanal and HNE, compared with the more
hydrophilic short-chain aldehydes acetaldehyde and acrolein, are
poorly scavenged by GSH. These data indicate that the lipophilic
aldehydes are not readily available to interact with the hydrophilic
aldehyde scavenger GSH, suggesting that long-chain aldehydes are
sequestered into a relatively hydrophobic plasma environment (eg, HDL)
that permits limited access to GSH.
DTNB Protects LCAT From Inhibition by Acrolein
The apparent sensitivity of LCAT to inactivation by aldehydes is
most likely related to its possession of two cysteine residues near its
active site. To test this hypothesis, we derivatized plasma LCAT with
the thiol-specific reversible LCAT inhibitor DTNB
before aldehyde exposure. Since LCAT was more sensitive to inactivation
by acrolein than was acetaldehyde, we used lower concentrations of
acrolein (0.164 to 1.31 mmol/L) than of acetaldehyde (2.63 to 21.0
mmol/L) in these experiments. The results of these studies are shown in
Fig 7. Blocking the free cysteine residues of LCAT
before acrolein exposure with DTNB resulted in almost complete
protection of the enzyme from inactivation (Fig 7). DTNB-treated plasma
exposed to 1.31 mmol/L acrolein experienced a 25% reduction in
activity, whereas untreated plasma exposed to the same acrolein
concentration lost more than 75% of its LCAT activity. In contrast,
DTNB was unable to protect LCAT from inhibition by acetaldehyde (Fig 7). These data suggest that of the two aldehydes examined, only
acrolein influences LCAT activity by interacting with the two free
cysteines of the enzyme.
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) and plasma treated with DTNB
before aldehyde exposure (
). Control plasma, without other
additions except PBS, was treated identically to experimental plasma.
The concentrations of the two aldehydes differ for these experiments
because LCAT is considerably more sensitive to inhibition by acrolein
than acetaldehyde (see Fig 2 |
Discussion |
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The present studies suggest that aldehydes that can be generated in vivo22 23 can modify HDL structure and inhibit LCAT activity. The aldehyde concentrations required for LCAT inhibition are generally less than those required to induce changes in HDL charge and structure. Moreover, the more effective a specific aldehyde is at inhibiting LCAT, the more effective it is at modifying HDL charge and cross-linking HDL apolipoproteins. Of the aldehydes examined, the
,ß-unsaturated aldehydes acrolein and HNE are
the most potent inhibitors of LCAT and cross-linkers of
HDL. In vivo, it is likely that aldehyde concentrations become elevated only in the interstitial fluid within focal areas in the extravascular space. Indeed, the presence of aldehydes and aldehyde-modified LDL (MDA and HNE) has been demonstrated in the atherosclerotic lesions of humans and rabbits.7 10 11 Since interstitial fluid is an ultrafiltrate of plasma, possessing fewer plasma proteins and lipoproteins, HDL and LCAT may be more susceptible to modification by aldehydes in this milieu than in plasma. However, because interstitial fluid is difficult to obtain, we substituted plasma for this fluid in our experiments. It should also be noted that the aldehyde concentrations used to modify plasma in the present study are similar to those used by others to modify ultracentrifugally isolated LDL and HDL.23 24 29 41
Aldehyde-induced protein modification involves the interaction of a
nucleophilic amino acid functional group (eg, primary amine or thiol)
with the aldehyde's carbonyl carbon.39 Subsequently, the
reversibly derivatized protein can condense with another reactive amino
acid residue (on the same protein or on a neighboring protein) to form
a more stable cross-linked product. The chemistry associated
with ,ß-unsaturated aldehydes is more complex than that
associated with saturated aldehydes, since the point of nucleophilic
attack may change from the carbonyl group to the carbon-carbon
double bond.39 40 Hence, ,ß-unsaturated
aldehydes, such as acrolein and HNE, may participate in more reactions
than the saturated aldehydes, acetaldehyde and hexanal, and they are
more likely to form stable adducts.39
Recent site-directed mutagenesis studies have shown that
thiol-specific reagents (eg, DTNB) inhibit LCAT by covalently
attaching to two free cysteine residues near the active site of
LCAT.42 43 Since ,ß-unsaturated aldehydes are
known to react more readily with sulfhydryl groups than saturated
aldehydes39 40 and since they are also more effective
inhibitors of LCAT, we thought it likely that aldehydes
inhibit LCAT by forming adducts with the free cysteine residues of the
enzyme. We tested this hypothesis by reversibly derivatizing the free
cysteines of LCAT with DTNB32 before aldehyde exposure.
This cysteine-blocking procedure was extremely effective at
preventing the ,ß-unsaturated aldehyde, acrolein, from
inhibiting LCAT. Although the enzyme was not completely protected, our
data suggest that >70% of the effects of acrolein, at concentrations
<1.3 mmol/L, could be attributed to cysteine modification. In
contrast, LCAT inhibition induced by the short-chain saturated
aldehyde, acetaldehyde, was completely unaffected by derivatization of
the free cysteines of LCAT with DTNB before acetaldehyde exposure.
Hence, acetaldehyde affects LCAT activity by a mechanism independent of
cysteine modification.
Since the reactivity of aldehydes to amino acid residues on proteins
relates to the specific functional group and the microenvironment in
which that functional group exists, it is difficult to predict which of
the potentially reactive amino acid residues within a protein will form
adducts with specific aldehydes. Although, as discussed above, it is
likely that the two free cysteine residues of LCAT are involved in its
inactivation by ,ß-unsaturated aldehydes, other mechanisms are
obviously involved. The catalytic site of LCAT is thought to consist of
a Ser-His-Asp "catalytic triad."44 Recently, Uchida
and Stadtman45 demonstrated that the histidine residues of
proteins are important targets for modification by HNE. They suggest
that the modification involves a Michael-type reaction between the
,ß-unsaturated bond of HNE and the imidazole nitrogen atom of
histidine.45 The formation of such an adduct with the
reactive histidine residue at the catalytic site of LCAT would most
certainly result in the loss of enzyme activity. In addition, one can
speculate that any aldehyde-induced modification of LCAT capable of
altering the charge properties and/or conformation of the enzyme (eg,
formation of lysine adducts, which are likely to be involved in the
inhibition of LCAT by saturated aldehydes) may affect substrate or
cofactor interactions.
In addition to inactivating LCAT, aldehydes also modified HDL protein structure, as reflected by cross-linking of apo A-I and apo A-II. The mechanisms for aldehyde-induced apolipoprotein modification are the same as those described for LCAT except that neither apo A-I nor apo A-II possesses free cysteine residues. The aldehyde concentrations required to modify HDL charge and cross-link apo A-I and A-II were consistently greater than those required to inhibit LCAT. This apparent difference in sensitivity to aldehyde modification between LCAT and the apolipoproteins of HDL may, as suggested above, reflect intrinsic differences between proteins. It should be emphasized, however, that changes in HDL structure rather than changes in HDL function were monitored in these experiments. HDL functional properties may therefore be affected by aldehydes before gross changes in protein structure are observed. It has been reported recently that MDA-treated HDLs are poor substrates for LCAT.29 Of considerable importance is the fact that these substrate effects on LCAT are quantifiable before the appearance of cross-linked forms of HDL. Similarly, we have found (J.Y.T. and M.R.M., unpublished observations) that ultracentrifugally isolated HDLs treated with either acrolein or HNE lose their ability to activate LCAT. This effect is detectable before the appearance of cross-linked forms of apo A-I and A-II.
Functional properties of HDL, other than LCAT activation, may also be impaired by cross-linking of the apolipoproteins of HDL. The ability of HDL3 to facilitate cholesterol efflux from macrophages and fibroblasts has been reported to be impaired in HDL3 modified by exposure to copper30 46 or MDA.30 Both treatments induce cross-linking of HDL-associated apolipoproteins. In contrast, apolipoproteins cross-linked on HDL3 by exposure to peroxidase-generated tyrosyl radicals appear to enhance the ability of HDL to facilitate cholesterol efflux.47 Despite some obvious differences in the manner in which these studies were performed and the extent of cross-linking, the disparate results are difficult to reconcile. It is possible, however, that the different cross-linking procedures produce different intramolecular and intermolecular linkages; proteins linked by one mechanism may have one effect and proteins linked by another mechanism may have a very different effect.
Relatively little is known concerning the in vivo events leading to the initiation of lipoprotein peroxidation. Whatever the mechanism, it is likely that both HDL and LDL are affected. Bowry et al27 recently reported that HDLs are the principal vehicle for circulating plasma lipid hydroperoxides and suggest that HDL lipids may be more easily peroxidized than those in LDL. It has also been suggested that lipid peroxidation products generated on LDL may transfer to HDL.28 Recent work from our laboratory is in agreement with this speculation. We have shown48 that copper-oxidized LDL (possessing thiobarbituric acid–reactive substances as low as 3 nmol/mg LDL protein) added back to the d>1.063-g/mL fraction of plasma containing both LCAT and HDL inactivates LCAT and cross-links HDL apolipoproteins. If, as suggested by these studies, HDL accumulates oxidized lipids in vivo capable of inhibiting LCAT and modifying HDL structure, it is likely that at least some of these effects are mediated by aldehydic breakdown products of lipid hydroperoxides. Lipophilic unsaturated aldehydes such as HNE may be particularly effective modifiers of LCAT structure and function because of their reactivity toward free thiols and likely sequestration by HDL in interstitial fluid.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This research was supported by funds provided by the Cigarette and Tobacco Surtax Fund of the state of California through the Tobacco-Related Disease Research Program of the University of California grant 2RT95 and National Institutes of Health Program Project grant HL-18574. Research was conducted at Lawrence Berkeley Laboratory (Department of Energy contract DE-AC03-76SF00098), University of California, Berkeley. We wish to thank Laura Knoff and Jan Selmek for their excellent technical assistance.
Received February 12, 1995; accepted July 18, 1995.
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