© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Oxidation of Apolipoprotein B-100 in Circulating LDL Is Related to LDL Residence Time
In Vivo Insights From Stable-Isotope Studies
From the Institute and Polyclinic of Clinical Metabolic Research (J.P., U.J.) and the Institute of Clinical Chemistry and Laboratory Medicine (P.L.), Medical Faculty, Technical University Dresden, Dresden, Germany.
Correspondence to Dr Jens Pietzsch, Institute and Polyclinic of Clinical Metabolic Research, Medical Faculty ‘Carl Gustav Carus,’ Technical University, Fetscherstrasse 74, D-01307 Dresden, Germany. E-mail julius{at}rcs.urz.tu-dresden.de
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Abstract |
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Abstract—5-Hydroxy-2-aminovaleric acid (HAVA) has been suggested to be a specific marker of oxidation of apolipoprotein (apo) B-100 proline (Pro) and arginine (Arg) side-chain residues in low density lipoprotein (LDL) in vitro. Here we describe the application of sensitive mass spectrometric techniques to the characterization of Pro/Arg-modified apoB-100 in LDL1 (Sf 7 to 12) and LDL2 (Sf 0 to 7) in vivo. We studied 7 subjects with familial defective apoB-100 (FDB) and 8 normolipidemic controls. In FDB subjects, the presence of a mutant apoB-100 (FDB3500Q) in LDL markedly reduced its affinity for the LDL receptor, leading to increased residence times (RTs) of LDL1 (65±21 versus 32±12 hours, P<0.005) and LDL2 (230±40 versus 53±7 hours, P<0.001) when compared with controls, as determined by stable-isotope turnover studies. LDL1 HAVA content was not different between the groups (FDB, 0.004±0.001 mol/mol apoB-100 versus controls, 0.003±0.001 mol/mol apoB-100, P=0.200). LDL2 HAVA content was higher in FDB subjects (0.374±0.088 versus 0.013±0.002 mol/mol apoB-100, P<0.001). In both groups, LDL2 HAVA was positively associated with LDL2 RT (FDB, r=0.893, P=0.003; controls, r=0.976, P=0.000) and negatively correlated with LDL2
-tocopherol content (FDB,
r=-0.929, P=0.003; controls,
r=-0.903, P=0.002). No significant correlations
could be found between LDL1 HAVA,
LDL1 RT, and -tocopherol,
respectively. The low LDL1 HAVA content observed
in both FDB and control groups was thought to be due to the relatively
lower RT as well as the higher -tocopherol content of
these lipoproteins. In contrast, LDL2 seemed to
be strongly prone to direct oxidation of apoB-100 in vivo. The longer
these particles linger in the circulation, the more apoB-100 Pro/Arg
residues become modified.
Key Words: familial defective apolipoprotein B-100 • lipoproteins • residence time • oxidation • atherosclerosis
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Introduction |
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Oxidative modification of LDL lipids and apoB-100 by reactive oxygen species (ROS) is widely regarded as a crucial event in atherogenesis.1 2 ApoB-100 modifications, eg, binding of lipid peroxidation products or direct oxidation of amino acid side-chain residues, are thought to finally result in the formation of new epitopes that are specifically recognized by scavenger receptors.1 2 However, despite convincing evidence from in vitro and animal experiments, data concerning the role of oxidized apoB-100 in the development of atherosclerosis in humans in vivo are scarce. One reason for this is the shortage of sensitive and specific methods for direct measurement of oxidized apoB-100 in circulating LDL. Recent studies have described the determination of circulating autoantibodies to oxidized LDL, the measurement of specific stable oxidation products of LDL apoB-100 isolated from human atherosclerotic tissue (eg, 3-chlorotyrosine or nitrotyrosine), or the measurement of stable oxidation products of tyrosine, valine, lysine, and phenylalanine side-chain residues of total LDL protein obtained from human plasma.3 4 5 More recently, the oxidation of LDL apoB-100 proline (Pro) and arginine (Arg) residues primarily to
-glutamyl semialdehyde, which by
reduction forms 5-hydroxy-2-aminovaleric acid (HAVA), has been measured
in vitro and in normolipidemic subjects in vivo.6 However,
the value of HAVA as a specific marker of LDL apoB-100 oxidation under
pathophysiological conditions such as
hypercholesterolemia has not been established.
Oxidative damage of LDL apoB-100 is believed to substantially occur in
the subendothelial space of the vessel
wall.1 During circulation, LDL particles enter and
reemerge from the subendothelium.2 7 Under
hypercholesterolemic conditions, eg, in subjects with
familial defective apoB-100 (FDB), the time needed to remove LDL from
the circulation is drastically increased.8 This should
favor an increase in the number of LDL particles that are exposed to
the subendothelium and in the duration of LDL apoB-100
exposure to ROS. Furthermore, compositional changes in LDL particles
possibly result in different exposures of both lipids and apoB-100 to
ROS.1 7 9 Thus, individuals with
hypercholesterolemia not only possess more
circulating LDL but also have older, modified LDL. In this context, we
hypothesized that the longer LDL particles linger in the circulation,
the more Pro and Arg residues of apoB-100 should be modified. To prove
this hypothesis, the present study combined specific and sensitive
gas chromatography–mass spectrometry (GC-MS)
methodologies to measure both HAVA formation and retention times of
native LDL in vivo in FDB subjects and normolipidemic controls. |
Methods |
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Subjects
Seven subjects with heterozygous FDB (3 men and 4 women; 21 to 63 years old) and 8 normolipidemic control subjects (4 men and 4 women; 20 to 51 years old) volunteered for the study. All subjects were nonsmokers and were free of renal, hepatic, hematological, and thyroid abnormalities, and all medications known to affect lipid levels were discontinued at least 6 weeks before the study. No subject was taking antioxidants such as probucol and vitamins A or E. All subjects were normoglycemic. All subjects gave informed, written consent; ethical approval was granted by the local ethics committee. Fasting blood samples were collected into tubes containing EDTA at a final concentration of 0.1%. The blood was centrifuged at 4°C (2000g for 10 minutes) to separate cells from plasma. Blood plasma and LDL samples were all processed in subdued light to prevent any photooxidation of LDL. All buffers and solutions were degassed and stored under argon. Buoyant LDL1 (Sf 7 to 12) and small, dense LDL2 (Sf 0 to 7) were isolated from plasma by a combination of both cumulative and sequential gradient ultracentrifugation techniques as previously described.8 Total LDL protein was determined by the bicinchoninic acid protein assay (Pierce) with bovine serum albumin as the protein standard. LDL apoB-100 was measured by immunoelectrophoresis (Sebia). LDL cholesterol was determined enzymatically by using CHOD-PAP test kits (Roche). The total carbonyl group content in LDL apoB-100 and LDL
-tocopherol content were measured as described elsewhere
and are expressed as mol/mol of apoB-100.6 9 10
Determination of HAVA
Delipidation of LDL, formation of HAVA by reduction of
-glutamyl semialdehyde with NaBH4, and
enzymatic hydrolysis of apoB-100 with nonspecific bacterial protease
type XIV (Sigma Chemical Co) were performed as previously
described.6 The free amino acids were isolated from
protein hydrolysates, derivatized to their
N(O)-ethoxycarbonyl ethyl ester derivatives, and
analyzed by electron-impact ionization GC-MS by following the
protocol described elsewhere.6 11 LDL HAVA content is
expressed as mol/mol of apoB-100. The intra-assay coefficient of
variation was <4.5%,and the interassay coefficient of variation,
<6.1%.
Determination of LDL ApoB-100 Residence Time
The determination of LDL apoB-100 residence time (RT) in FDB and
control subjects has been described elsewhere.8 In brief,
the stable-isotope tracers used were
L-[ring-13C6]phenylalanine
or L-[5,5,5-2H3]leucine.
After administration of a priming bolus of 550 µg/kg of
[13C6]phenylalanine
([2H3]leucine data in
parentheses; 655 µg/kg), a constant infusion of 12 µg ·
kg-1 · min-1 (16
µg · kg-1 ·
min-1) was continued for 12 hours. Blood samples
were obtained before the priming bolus; at 10-minute intervals for 2
hours; and after 2, 2.5, 3, 3.5, 4, 5, 6, 9, 10, 11, 12, 24, 48, and 72
hours. ApoB-100 of LDL subfractions was separated by preparative
polyacrylamide gel electrophoresis. The stained apoB-100 bands
were excised from gels and hydrolyzed. The free amino acids were
isolated from apoB-100 hydrolysates by cation-exchange
chromatography and then derivatized, and isotopic
enrichment was determined by GC-MS. The kinetic parameters
of LDL apoB-100 metabolism were estimated by
multicompartmental analysis using the
SAAM (simulation analysis and modeling,
version 31) software package as previously
published.8 After fitting the model to the tracer
data, LDL apoB-100 fractional catabolic rates and RTs were determined
with reasonable certainty on the basis of the fractional standard
deviations of the model parameter estimates.8
In our studies, LDLs were fractionated into 2 subclasses of particles:
"buoyant" LDL1 and smaller, more dense
LDL2. Here, the enrichment curves clearly
indicated that the labeling of LDL1 preceded that
of LDL2; hence, they were modeled as precursor
and product, respectively.8
Statistical Analysis
Descriptive data were expressed as arithmetic means±SDs.
Statistical analyses (Mann-Whitney tests, Spearman rank
correlation analysis) were calculated by using the
SPSS 9.0 software package.
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Results |
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FDB patients had significantly higher levels of plasma total cholesterol, plasma apoB-100, and cholesterol and apoB-100 levels of LDL1 and LDL2 (Tables I
and II), whereas the concentrations of plasma
triglycerides and lipoprotein constituents of VLDL, IDL,
and HDL particles were not different between the 2 groups (statistical
results not shown in detail).8 The mean lipoprotein mass
composition of LDL1 and
LDL2 particles, ie, the mass percentage of
cholesterol and apoB-100 of total lipoprotein mass, did not
differ between the 2 groups. Furthermore, the
-tocopherol content in LDL1 and
LDL2 particles was similar in the 2 groups.
However, in both groups, the -tocopherol content in
LDL1 was significantly higher when compared with
that in LDL2 (P=0.000). The
cholesterol-rich, buoyant LDL1
particles contained 
5 molecules of -tocopherol per
particle, whereas the cholesterol-poor, dense
LDL2 particles contained 1 molecule of
-tocopherol per particle (Table II). The HAVA content in total LDL
(LDL1 plus LDL2) was
significantly higher in FDB subjects when compared with controls
(0.063±0.020 versus 0.004±0.001 mol/mol apoB-100,
P<0.001). LDL1 HAVA content
showed no differences between the 2 groups. LDL2
particles in FDB contained significantly more HAVA molecules than did
LDL2 in controls. Furthermore, the HAVA content
in LDL2 was higher when compared with
LDL1 within and between the 2 groups (Table III). The apoB-100 carbonyl group content in total LDL was
somewhat but not significantly higher in FDB (0.21±0.08 versus
0.18±0.03 mol/mol apoB-100, P=0.064). The
LDL1 apoB-100 carbonyl group content showed no
differences between the 2 groups (0.05±0.02 versus 0.06±0.07 mol/mol
apoB-100, P=0.090), whereas LDL2
particles in FDB contained significantly more carbonyl groups than did
LDL2 in controls (0.16±0.04 versus 0.12±0.03,
P<0.01). The mean RT of LDL subfractions (reciprocal of
fractional catabolic rate at steady state) derived by
multicompartmental analysis is presented in Table III. The other kinetic parameters of
apoB-containing lipoproteins in the subjects studied have been
published previously and are not shown in detail.8
The mean RT of total LDL (LDL1 plus
LDL2) apoB-100 was >3-fold higher in FDB when
compared with controls (147.2±19.3 versus 42.2±3.7 hours;
P<0.001). In FDB, the mean RT of LDL1
and LDL2 particles was increased 2-fold and
>4-fold, respectively, when compared with controls (Table III). In both groups, significant, positive correlations were
found between LDL2 HAVA and
LDL2 RT (Figure I).
In addition, in both groups, LDL2 HAVA content
was significantly correlated in a negative manner with
LDL2 -tocopherol (Figure
II) and positively with
LDL2 apoB-100 carbonyl group content (FDB,
r=0.764, P=0.012; controls, r=0.508,
P=0.030). No significant correlations were found between
LDL1 HAVA, LDL1 RT,
LDL1 -tocopherol, and
LDL1 apoB-100 carbonyl group content,
respectively.
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Discussion |
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-Glutamyl semialdehyde is a primary oxidation product of
both Pro and Arg side-chain residues.5 12 13 By reduction
with NaBH4, -glutamyl semialdehyde forms
HAVA.6 12 13 Recently, HAVA has been shown to be a
specific marker for apoB-100 oxidation in vitro and in normolipidemic
subjects in vivo.6 In that former study, the level of HAVA
demonstrated in native total LDL obtained from 10 normolipidemic, young
male volunteers was 0.012±0.004 mol/mol apoB-100 (0.4:10 000,
Pro/Arg). The present work for the first time reports experiments
with HAVA as a highly specific and sensitive marker of direct apoB-100
oxidation of circulating human LDL subfractions under
pathophysiological conditions. Therefore, as a
model, patients with heterozygous FDB showing a moderate to severe
hypercholesterolemia were studied. The risk for
the development of premature ischemic heart disease is strongly
increased in FDB.14 The apoB-100 defect (Arg3500Gln)
primarily affects the fractional catabolism of LDL.8 15
The in vivo consequence of this has been shown to be a 2-fold higher RT
of buoyant LDL1 and a >4-fold higher RT of small
LDL2 in FDB when compared with
controls.8 The longer the RT of LDL, the longer is the
exposure of its apoB-100 moiety to the attack of ROS. ApoB-100 consists
of 4563 amino acids and has a molecular weight of 516 000 (minus the
carbohydrate content).16 ApoB-100 contains 170 Pro and 148
Arg residues that are partially susceptible to direct, oxidative damage
in vivo.6 HAVA is suggested to be formed by
metal-catalyzed oxidation processes.6 Apparently,
Cu2+ or Fe2+ bind to
discrete sites of apoB-100 and form centers for repeated radical
production. The exact number of such binding sites is not
known, and values ranging from 3 to 12 have been reported by
others.9 17 However, participation of other
ROS-generating processes, eg, myeloperoxidase reaction, in the
formation of HAVA is unknown. The present study shows higher HAVA
levels in total LDL in FDB subjects when compared with controls
(1.97:10 000 versus 0.13:10 000 Pro/Arg, P<0.01). In
controls, LDL2 HAVA was higher when compared with
LDL1, but in total it did not exceed the range
demonstrated in healthy men elsewhere.6 In FDB,
apoB-100 of circulating LDL2 contained
significantly higher amounts of modified Pro/Arg residues when compared
with controls. Here, the level of HAVA amounted to 12:10 000
Pro/Arg residues. There exists a strong association between the
extremely higher RT of LDL2 and the oxidative
modification of apoB-100 Pro/Arg in FDB. These findings are
consistent with the increment in nonspecific carbonyl group
content in LDL2 apoB-100 in FDB. In contrast,
HAVA levels in LDL1 particles were not increased
in FDB, and no associations could be found with the RT of
LDL1. In addition, qualitative changes in the LDL
particles could render them more or less prone to oxidation. For
instance, LDL -tocopherol molecules are supposed to be
good competitive substrates for oxidative attack.7 9 In
hypercholesterolemic subjects, LDL
-tocopherol is suggested to be a predictor of LDL
oxidizability.9 18 In the present study, LDL
-tocopherol levels were not different between the groups
but were significantly higher in LDL1 particles
when compared with LDL2 particles.
LDL2 -tocopherol showed a strong,
negative association with LDL2 HAVA. Although
there is no significant relationship between LDL1
apoB-100 modification and LDL1
-tocopherol, the higher content of the antioxidant could
provide an explanation for the lower extent of HAVA formation in
LDL1. This is consistent with data
published by others.7 9 17 18 In conclusion, because HAVA
is not a normal constituent of human apolipoproteins, the overall yield
of HAVA that has been found in LDL2 apoB-100 is
remarkably high in FDB and indicates that LDL apoB-100 Pro/Arg residues
are good targets for oxidative attack under present
pathophysiological conditions. Our data suggest
that oxidative damage of a particularly small, more dense,
-tocopherol–poor, and "aged" LDL entity both in
blood and in the subendothelium may be an important
mechanism underlying the premature ischemic heart disease in
FDB. However, additional work is needed to understand the specific
consequences of -glutamyl semialdehyde formation for the
metabolic fate of apoB-containing lipoproteins in vivo.
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Acknowledgments |
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The published work is part of the thesis of J.P. for his postgraduate study of toxicology and environmental medicine at the Institute of Legal Medicine at the Leipzig University, Germany.
Received June 28, 2000; accepted August 16, 2000.
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