Platelet-Activating Factor Acetylhydrolase Is Mainly Associated
With Electronegative Low-Density Lipoprotein Subfraction
Sonia Benítez, PhD*; José Luis Sánchez-Quesada, PhD*; Vicent Ribas, BSc; Oscar Jorba, BSc;
Francisco Blanco-Vaca, MD, PhD; Francesc González-Sastre, MD, PhD; Jordi Ordóñez-Llanos, MD, PhD
Downloaded from http://circ.ahajournals.org/ by guest on June 14, 2017
Background—Electronegative LDL [LDL(⫺)], a modified subfraction of LDL present in plasma, induces the release of
interleukin-8 and monocyte chemotactic protein-1 from cultured endothelial cells.
Methods and Results—We demonstrate that platelet-activating factor acetylhydrolase (PAF-AH) is mainly associated with
LDL(⫺). LDL(⫺) had 5-fold higher PAF-AH activity than the nonelectronegative LDL subfraction [LDL(⫹)] in both
normolipemic and familial hypercholesterolemic subjects. Western blot analysis after SDS-PAGE confirmed these
results, because a single band of 44 kDa corresponding to PAF-AH appeared in LDL(⫺) but not in LDL(⫹).
Nondenaturing polyacrylamide gradient gel electrophoresis demonstrated that PAF-AH was bound to LDL(⫺)
regardless of LDL size. In accordance with the above findings, nonesterified fatty acids, a cleavage product of PAF-AH,
were increased in LDL(⫺) compared with LDL(⫹).
Conclusions—The high PAF-AH activity observed in LDL(⫺) could be related to the proinflammatory activity of these
lipoproteins toward cultured endothelial cells. (Circulation. 2003;108:92-96.)
Key Words: lipoproteins 䡲 inflammation 䡲 fatty acids
P
with the most remarkable difference being an increased
NEFA content in LDL(⫺) compared with native LDL.7,8 The
aim of the present work was to study whether the origin of
increased NEFA in LDL(⫺) could be related to LDLassociated PLA2 activity.
latelet-activating factor acetylhydrolase (PAF-AH) is a
serine lipase that hydrolyzes the sn-2 ester bond of PAF
and attenuates its high biological activity.1 PAF-AH circulates mainly bound to LDL and, owing to its phospholipase
A2 (PLA2) activity, is also known as lipoprotein-associated
PLA2.2 Growing evidence suggests that PAF-AH is involved
in atherosclerosis, although controversy exists regarding its
proinflammatory or antiatherogenic properties. Its capacity to
hydrolyze PAF and other PAF-like lipids suggests an antiatherogenic role.1 However, hydrolysis of PAF-like molecules produces lysophosphatidylcholine (LPC) and oxidized
or fractionated nonesterified fatty acids (NEFA), which are
also well-known inflammation mediators.3 Furthermore,
plasma levels of lipoprotein-associated PLA2 appeared to be
an independent risk factor for coronary artery disease in
normolipemic and hypercholesterolemic subjects.4,5 The observation that PAF-AH inhibition in rabbits slows atherosclerosis progression provided further evidence of the relationship between PAF-AH and atherogenesis.6 Electronegative
LDL [LDL(⫺)] is a subfraction of LDL present in plasma that
induces the release of interleukin-8 and monocyte chemotactic protein-17,8 and is cytotoxic in cultured human endothelial
cells.9,10 The mechanism by which LDL(⫺) induces chemokine release remains unknown, although it could be related to
some compositional differences compared with native LDL,
Methods
Lipoprotein Isolation
Plasma samples were obtained from normolipemic (NL) or familial
hypercholesterolemic (FH) subjects, as described previously.8 The
study was approved by the Ethics Committee of the hospital, and all
subjects gave informed consent. Total LDL (1.019 to 1.050 g/mL)
was isolated by sequential ultracentrifugation and fractionated into
electropositive [LDL(⫹); elution at 0.22 mol/L NaCl] and LDL(⫺)
(elution at 0.50 mol/L NaCl) by anion-exchange chromatography.7
This density range was chosen to avoid contamination with lipoprotein(a) [Lp(a)], which is known to have a high amount of PAF-AH.11
Composition of LDL fractions, including total and free cholesterol,
triglyceride, phospholipid, apolipoprotein (apo) B, and NEFA, was
determined as described previously.7
Because ultracentrifugation was reported to promote the displacement of PAF-AH from LDL to HDL or lipoprotein-deficient serum,12 an alternative method to ultracentrifugation was used. Total
LDL from human NL plasma was isolated by gel filtration chromatography, as described previously.13 Briefly, 10 mL of plasma was
subjected to chromatography consecutively (1 mL in each chromatograph) in a Superose 6 column (Amersham Biosciences) and
collected in 1-mL fractions, and cholesterol, triglyceride, apoB, and
Received January 23, 2003; revision received March 25, 2003; accepted March 26, 2003.
From the Servei de Bioquímica and Institut de Recerca (S.B., J.L.S.-Q., V.R., O.J., F.B.-V., F.G.-S., J.O.-L.), Hospital de la Santa Creu i Sant Pau,
and Departament de Bioquímica i Biología Molecular (F.G.-S., J.O.-L.), Universitat Autònoma de Barcelona, Barcelona, Spain.
*Drs Benítez and Sánchez-Quesada contributed equally to this work.
Correspondence to Dr Jordi Ordóñez-Llanos, Servei de Bioquímica, Hospital de la Santa Creu i Sant Pau, C/Antoni María Claret 167, 08025 Barcelona,
Spain. E-mail [email protected]
© 2003 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000072791.40232.8F
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Benítez et al
PAF-AH and Electronegative LDL
93
TABLE 1. Composition and PAF-AH Activity of LDL Subfractions (nⴝ8) Isolated
From NL and FH Subjects
NL Subjects
FH Subjects
LDL(⫹)
LDL(⫺)
LDL(⫹)
LDL(⫺)
Total cholesterol, % of LDL mass
40.5⫾1.0
40.7⫾0.5
42.3⫾0.8
43.3⫾0.3*
Free cholesterol, % of LDL mass
11.7⫾0.9
12.2⫾1.0
11.8⫾0.3
11.9⫾0.6
Triglycerides, % of LDL mass
7.3⫾1.5
Phospholipids, % of LDL mass
25.9⫾2.6
25.9⫾2.7
25.8⫾2.3
25.4⫾1.4
ApoB-100, % of LDL mass
26.3⫾1.6
24.6⫾1.9*
25.1⫾2.5
22.6⫾0.9*
NEFA, mol/mol apoB
PAF-AH activity, ␮mol 䡠 min⫺1 䡠 mL⫺1
8.7⫾2.1*
6.7⫾1.2
8.6⫾1.0*
8.9⫾1.9
16.8⫾6.2*
7.6⫾2.3
11.8⫾3.7*
1.17⫾0.55
6.71⫾1.29*
1.13⫾0.34
5.08⫾3.01*
*P⬍0.05 vs LDL(⫹).
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apoA-I were measured. Fraction 11, corresponding to the purest LDL
fraction (apoB⫽0.20 g/L, apoA-I⫽0.00 g/L, cholesterol⫽0.68 mmol/L, triglyceride⫽0.07 mmol/L, total volume 10 mL),
was subfractionated in LDL(⫹) and LDL(⫺) by anion-exchange
chromatography as described above. LDL(⫹) and LDL(⫺) subfractions were concentrated with an Amicon ultrafiltration concentrator.
PAF-AH Activity
A commercial colorimetric assay (Cayman Chemical) was used. This
method uses 2-thio-PAF as a substrate and purified human PAF-AH
as a positive control. LDL fractions (10 ␮L of LDL at 0.25 g/L apoB,
in Tris 20 mmol/L, EDTA 1 mmol/L, pH 7.4) were incubated with
2-thio-PAF and 5,5⬘-dithio-bis(2-nitrobenzoic acid) as indicated by
the manufacturer. Absorbance was measured at 414 nm at increased
times in a microtiter plate, and the slope was used to calculate
PAF-AH activity (expressed as micromoles per minute per
milliliter).
Lecithin:Cholesterol Acyltransferase Activity
Lecithin:cholesterol acyltransferase activity in both LDL fractions
was determined by measuring the esterification of labeled free
cholesterol with liposomes formed by 3H-free cholesterol/phosphatidylcholine/cholate, as described previously.14
Sodium Dodecyl Sulfate–Polyacrylamide
Gel Electrophoresis
Commercial 3% to 8% polyacrylamide gels (Novex) were used. Gels
were prerun for 30 minutes with Tris-glycine buffer (Tris
50 mmol/L, glycine 384 mmol/L, pH 8.3) containing 0.1% (wt/vol)
SDS. LDL subfractions (5 ␮g of apoB) diluted vol/vol in Laemmli
sample buffer without 2-mercaptoethanol (BioRad) were electrophoresed for 10 minutes at 25 V, 10 minutes at 50 V, and 1 hour at 100
V. Rainbow marker (Amersham) was used as the molecular weight
standard.
Nondenaturing Polyacrylamide Gradient
Gel Electrophoresis
Nondenaturing polyacrylamide gradient gel electrophoresis (GGE)
gels (2% to 16% polyacrylamide) were made as described previously,15 and electrophoresis developed for 30 minutes at 30 V, 30
minutes at 70 V, and 12 hours at 100 V. Five micrograms of apoB
of each LDL subfraction were applied in each lane. A mixture of
plasma containing 4 LDL bands of known size was used as
standard.15
Western Blot Analysis
Proteins were transferred to a polyvinylidene difluoride (PVDF)
membrane (Immun-blot PVDF, BioRad) in a Mini Trans-blot electrophoretic transfer cell (BioRad) for 1 hour at 30 V with Tris
25 mmol/L, glycine 192 mol/L, methanol 20%, and SDS 0.1%
buffer. PVDF membranes were blocked overnight with Tris-HCl
10 mmol/L, NaCl 150 mmol/L, pH 7.4 buffer (TBS) containing 5%
nonfat dry milk, incubated for 1.5 hours with PAF-AH or secretory
PLA2 (sPLA2) polyclonal antiserum (Cayman Chemical) diluted
1/1000 with TBS containing 2.5% nonfat dry milk, washed 4 times
with TBS containing 0.1% Tween 20 (TTBS), and then incubated for
1.5 hours with the secondary antibody labeled with peroxidase
(dilution 1/5000, goat anti-rabbit IgG, Sigma). After 4 washes with
TTBS, membranes were developed with a chemoluminescent substrate (Supersignal CL-HRP substrate system, Pierce).
Statistical Analysis
Results are expressed as mean⫾SD. Differences between groups
were tested with Wilcoxon t test (for paired data) and Mann-Whitney
U test (for unpaired data). A value of P⬍0.05 was considered
significant.
Results
The LDL(⫺) proportion was 8.0⫾2.3% in NL samples (n⫽8)
and 15.6⫾2.9% in FH samples (n⫽8; P⬍0.05). Composition
of LDL subfractions (Table 1) confirmed features observed
previously,7,8 including increased NEFA content in LDL(⫺)
compared with LDL(⫹). PAF-AH activity was approximately 5-fold higher in LDL(⫺) than in LDL(⫹) from both
NL and FH subjects (Table 1).
Western blot analysis confirmed that PAF-AH was preferably associated with LDL(⫺) (Figure 1). Western blot of
SDS-PAGE gels showed an intense band of 44 kDa in
LDL(⫺) from NL and FH, corresponding to PAF-AH. This
band was absent or very faint in LDL(⫹) fractions (Figure
1B). There is an apparent discrepancy between the 5-fold
enrichment of PAF-AH in LDL(⫺) measured by enzymatic
activity and Western blot that suggests greater enrichment.
However, these differences could be more apparent than real.
First, Western blot is not an appropriate method for quantification; thus, the absence of PAF-AH in LDL(⫹) observed
in some Western blots is probably due to lack of sensitivity.
The absolute amount of PAF-AH carried in LDL fractions is
unknown, but the molar ratio apoB/PAF-AH is clearly higher
than 1, even in LDL(⫺). Packard et al4 reported that normolipemic subjects have ⬇2 mg/L PAF-AH in plasma, in
contrast to ⬇1 g/L apoB. This indicates that the amount of
PAF-AH electrophoresed in each lane could be in the order of
nanograms. In the present experimental conditions, we observed that PAF-AH was detected in LDL(⫺) when at least 1
␮g of apoB was injected into the gel (Figure 2); because
Figure 1 shows 5 ␮g of apoB both in LDL(⫹) and LDL(⫺),
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Circulation
July 8, 2003
Figure 1. Representative SDS-PAGE and
Western blot analysis of LDL subfractions from NL and FH subjects. Five
micrograms of apoB were injected into
each lane and electrophoresis developed
as described in Methods. A, SDS-PAGE
stained with Coomassie Brilliant Blue; B,
Western blot revealed with anti-PAF-AH.
Mol.weight indicates molecular weight.
Downloaded from http://circ.ahajournals.org/ by guest on June 14, 2017
it is clear that a difference in PAF-AH from 5- to 6-fold
between LDL(⫹) and LDL(⫺) would result in the absence
[or the observation of very faint bands, as in LDL(⫹)] or the
presence of a well-defined band corresponding to PAF-AH
[as in LDL(⫺)].
Western blot of GGE demonstrated that PAF-AH was
bound to LDL(⫺), because PAF-AH bands resembled the
bands of LDL(⫺) observed in gels stained with Coomassie
Brilliant Blue (Figure 3). LDL(⫺) from FH subjects presented an intense band of greater than normal-sized LDL bands.
This band could be due to the presence of small aggregates or
to contamination with Lp(a). Evidence indicates that this
band was not Lp(a). First, the density range was chosen to
avoid contamination with Lp(a) in LDL. Second, Lp(a)
content in both LDL fractions, measured by immunoturbidimetry (Roche), was lower than 0.9% in LDL(⫹) and 2.1%
in LDL(⫺) in all LDL preparations, in accordance with
previous reports.7,8 Finally, the intensity of this band by
protein staining (Figure 3A) was much higher than when
revealed by Western blot (Figure 3B); this observation is
inconsistent with the finding that Lp(a) contains a 7-fold
higher amount of PAF-AH than LDL.11 Hence, we concluded
that this band of greater than normal-sized LDL was due to
small aggregates.
The possibility that PAF-AH was coisolated with LDL(⫺)
because of artifacts produced during ultracentrifugation was
tested by isolating total LDL from plasma by gel filtration
chromatography. Western blot in GGE (Figure 4) and
PAF-AH activity of LDL subfractions separated from total
LDL isolated by gel filtration showed a patent increase in
PAF-AH associated with both LDL fractions compared with
samples isolated by ultracentrifugation [PAF-AH activity
(␮mol · min⫺1 · mL⫺1) of total LDL isolated by chromatography, LDL(⫹)0.55, LDL(⫺)9.75; total LDL isolated by
ultracentrifugation, LDL(⫹) 0.35, LDL(⫺) 4.50], in accordance with previous reports.12 However, this decrease does
not modify the basic observation that most PAF-AH is bound
to LDL(⫺).
The presence in LDL fractions of other enzymes with PLA2
activity, such as sPLA2 and lecithin:cholesterol acyltransferase, was studied. sPLA2 was not observed in either
LDL(⫹) or LDL(⫺) by Western blot (data not shown).
Moreover, the presence of EDTA 1 mmol/L in LDL samples
during the activity assay also ruled out the possibility of
2-thio-PAF degradation being due to sPLA2, because this
enzyme needs calcium for its enzymatic activity. Lecithin:cholesterol acyltransferase activity was very low and equal
in both LDL fractions (0.03⫾0.01 nmol · h⫺1 · mL⫺1).
Preincubation of both LDL fractions with Pefabloc 500
␮mol/L (Sigma), a known specific inhibitor of PAF-AH,16
abolished the hydrolyzing activity of PAF-AH by up to 90%
(Table 2), which indicates that hydrolysis of thio-PAF was
specific for PAF-AH.
Discussion
Figure 2. Sensitivity of Western blot PAF-AH analysis. Increasing amounts of LDL(⫺) from NL subjects (1, 3, and 5 ␮g of
apoB) were electrophoresed in SDS-PAGE gels, transferred to
PVDF membranes, and revealed with anti-PAF-AH antibody, as
described in Methods.
PAF-AH has been proposed as a potential risk factor for
coronary artery disease, and its plasma concentration and
activity have been shown to be directly correlated with
plasma and LDL cholesterol.4,5 The present work demonstrates that LDL-borne PAF-AH is preferentially bound to
LDL(⫺). Increased PAF-AH activity associated with
LDL(⫺) in NL subjects concurs with reports of preferential
distribution of PAF-AH in dense LDL subfractions,17 because
LDL(⫺) from NL subjects has been reported to be more
abundant in dense subfractions.16,18 However, LDL(⫺) from
FH patients is abundant in light subfractions16; hence, the
Benítez et al
PAF-AH and Electronegative LDL
95
Figure 3. Representative GGE and Western blot analysis of LDL subfractions
from NL and FH subjects. Five micrograms of apoB were injected into each
lane and electrophoresis developed as
described in Methods. A, GGE stained
with Coomassie Brilliant Blue; B, Western blot revealed with anti-PAF-AH.
Downloaded from http://circ.ahajournals.org/ by guest on June 14, 2017
observation that PAF-AH was also preferentially carried in
LDL(⫺) from FH suggests that its association depends on
lipoprotein characteristics other than density or size.
Some concerns could be raised based on the possibility that
the predominant association of PAF-AH with LDL(⫺) is an
artifactual feature due to the methods used to isolate LDL(⫺).
It was described that ultracentrifugation results in the loss of
PAF-AH in LDL and a parallel enrichment of PAF-AH in
HDL and lipoprotein-deficient serum,12 but a redistribution of
PAF-AH from LDL(⫹) to LDL(⫺) is highly improbable
because both fractions are of very similar density. Nevertheless, we tested this possibility. Ultracentrifugation did not
modify the fact that most PAF-AH was bound to LDL(⫺), as
demonstrated by the experiment using gel filtration chromatography to isolate total LDL. Concerning the use of anionexchange chromatography to separate LDL(⫺) from
LDL(⫹), no alternative methods based on the electric charge
of molecules exist, because electrophoresis is an excellent
analytical technique but is not suitable as a preparative
method. However, several arguments indicate that it is very
improbable that anion-exchange chromatography promoted a
preferential association of PAF-AH with LDL(⫺). According
to previous reports by Stafforini and colleagues,19 it is
unlikely that PAF-AH leaves LDL as a consequence of a
relatively small change in ionic strength. Those authors
reported that the interaction between PAF-AH and LDL
involves protein-protein interactions between specific residues of PAF-AH and apoB.19 They also suggested that these
interactions were tight because PAF-AH was copurified with
a carboxyl-terminal fragment of apoB.20 In earlier works,21,22
these authors failed to isolate PAF-AH from LDL using
changes in the ionic composition and strength of the buffer
and had to use detergents (Tween 20) to separate LDL from
PAF-AH. In the same report, it was stated that PAF-AH
activity elutes in a DEAE-Sepharose column (an anionexchange resin similar to Q-Sepharose) at a lower ionic
strength than most of the LDL/VLDL precipitate injected,
which indicates that PAF-AH has a lower electronegativity
than most LDL. In a more recent work,23 in which CHAPS
was used instead of Tween 20 to isolate PAF-AH, it was
reported that PAF-AH elutes in a MonoQ column (which
contains the same quaternary amine as Q-Sepharose as active
group) at an ionic strength of 0.15 to 0.20 mol/L NaCl; hence,
PAF-AH alone would elute in the first step (0.22 mol/L NaCl)
of the stepwise gradient that we used to separate both LDL
fractions, ie, in the step containing LDL(⫹). These observations indicate that it is very unlikely that either ultracentrifugation or anion-exchange chromatography could induce an
artifactual binding of PAF-AH to LDL(⫺).
The association between LDL(⫺) and PAF-AH suggests
several implications concerning the atherogenicity of these
lipoproteins. The mechanisms by which LDL(⫺) induces
chemokine release are poorly understood. Although some
authors reported that LDL(⫺) contained increased oxidation
products,9,18,24 other investigators found no evidence of oxidative modification.7,8,10 The high activity of PAF-AH in
LDL(⫺) points to a possible role of this enzyme in the
inflammatory effect of LDL(⫺). PAF-AH activity generates
LPC and short-chain or oxidized NEFA. In accordance with
this, increased NEFA7,8 and LPC24 content was observed in
LDL(⫺). Both LPC and NEFA are known inflammation
mediators. LPC is chemotactic for monocytes,3,25 induces
apoptosis in endothelial and smooth muscle cells,26 and
TABLE 2. Effect of LDL Subfraction Preincubation With
the PAF-AH–Specific Inhibitor Pefabloc (500 ␮mol/L) on
2-thio-PAF Degradation
Control
Pefabloc
(500 ␮mol/L)
PMSF
(4 mmol/L)
NL-LDL(⫹)
0.50⫾0.14
0.18⫾0.11
0.30⫾0.14
NL-LDL(⫺)
5.82⫾0.95
0.20⫾0.14
0.08⫾0.04
FH-LDL(⫹)
0.80⫾0.28
0.28⫾0.11
0.20⫾0.07
FH-LDL(⫺)
4.48⫾0.67
0.10⫾0.07
0.10⫾0.07
PAF-AH Activity
(␮mol 䡠 min⫺1 䡠 mL⫺1)
Figure 4. Western blot of LDL subfractions from NL subjects.
Samples were electrophoresed in nondenaturing GGE, transferred to PVDF membranes, and revealed with anti-PAF-AH
antibody, as described in Methods. Three micrograms of apoB
were injected into each lane. In lanes 1 to 3, total LDL was isolated by gel filtration chromatography [1, total LDL; 2, LDL(⫹); 3,
LDL(⫺)]. In lanes 4 and 5, total LDL was isolated by ultracentrifugation [4, LDL(⫹); 5, LDL(⫺)].
The effect of PMSF (4 mmol/L), a general inhibitor of serine lipases, is also
shown. Results are mean of duplicates from 2 independent experiments.
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Circulation
July 8, 2003
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increases monocyte chemotactic protein-1 expression in endothelial cells.27 Oxidized NEFA is also a chemoattractant to
monocytes.3 The present findings suggest that chemokine
release induced by LDL(⫺) could be a consequence of the
high PAF-AH activity in LDL(⫺) through the production of
LPC, NEFA, or both.
This observation is consistent with a proinflammatory role
for PAF-AH, in accordance with its positive correlation with
plasma cholesterol and coronary artery disease risk.4,5 The
use of specific inhibitors also supports this possibility, because PAF-AH inhibition decreased monocyte chemotactic
activity3,23 and apoptosis26 induced by “in vitro”– oxidized
LDL and reduced atherosclerotic plaque development in a
3-month study in rabbits.6 However, the putative proinflammatory role of PAF-AH should be considered with caution,
because its anti-inflammatory role through the degradation of
PAF and PAF-like lipids is well established.1 The possibility
exists that the proinflammatory or anti-inflammatory role
depends on the lipoprotein carrying PAF-AH in plasma. A
protective role of PAF-AH associated with HDL has been
suggested,28,29 and the distribution between LDL and HDL
might be an important determinant of risk.30
In summary, we report that most PAF-AH activity is bound
to LDL(⫺) and suggest that this enzyme could be related to
the chemokine release induced by LDL(⫺) in endothelial
cells. Further studies are required to define the role of
LDL(⫺)-bound PAF-AH.
Acknowledgments
This work was supported by grants SAF2001/0480C02-01 to Dr
Ordóñez-Llanos and SAF1999/0104 to Dr Blanco-Vaca from the
Ministerio de Ciencia y Tecnología. V. Ribas is a recipient of a
predoctoral fellowship from Ministerio de Ciencia y Tecnología. The
authors are grateful to Christine O’Hara for editorial assistance.
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Platelet-Activating Factor Acetylhydrolase Is Mainly Associated With Electronegative
Low-Density Lipoprotein Subfraction
Sonia Benítez, José Luis Sánchez-Quesada, Vicent Ribas, Oscar Jorba, Francisco Blanco-Vaca,
Francesc González-Sastre and Jordi Ordóñez-Llanos
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Circulation. 2003;108:92-96; originally published online June 23, 2003;
doi: 10.1161/01.CIR.0000072791.40232.8F
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