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PHAGOCYTES
Defects in leukocyte-mediated initiation of lipid peroxidation in plasma as studied
in myeloperoxidase-deficient subjects: systematic identification of multiple
endogenous diffusible substrates for myeloperoxidase in plasma
Renliang Zhang, Zhongzhou Shen, William M. Nauseef, and Stanley L. Hazen
More than a decade ago it was demonstrated that neutrophil activation in
plasma results in the time-dependent formation of lipid hydroperoxides through
an unknown, ascorbate-sensitive pathway. It is now shown that the mechanism
involves myeloperoxidase (MPO)-dependent
use of multiple low-molecular–weight substrates in plasma, generating diffusible
oxidant species. Addition of activated human neutrophils (from healthy subjects)
to plasma (50%, vol/vol) resulted in the
peroxidation of endogenous plasma lipids by catalase-, heme poison-, and ascorbate-sensitive pathways, as assessed by
high-performance liquid chromatography
(HPLC) with on-line electrospray ionization tandem mass spectrometric analysis
of free and lipid-bound 9-HETE and
9-HODE. In marked contrast, neutrophils
isolated from multiple subjects with MPO
deficiency failed to initiate peroxidation
of plasma lipids, but they did so after
supplementation with isolated human MPO.
MPO-dependent use of a low-molecular–
weight substrate(s) in plasma for initiating lipid peroxidation was illustrated by
demonstrating that the filtrate of plasma
(10-kd MWt cutoff) could supply components required for low-density lipoprotein
lipid peroxidation in the presence of MPO
and H2O2. Subsequent HPLC fraction-
ation of plasma filtrate (10-kd MWt cutoff)
by sequential column chromatography
identified nitrite, tyrosine, and thiocyanate as major endogenous substrates
and 17␤-estradiol as a novel minor endogenous substrate in plasma for MPO in
promoting peroxidation of plasma lipids.
These results strongly suggest that the
MPO–H2O2 system of human leukocytes
serves as a physiological mechanism for
initiating lipid peroxidation in vivo. (Blood.
2002;99:1802-1810)
© 2002 by The American Society of Hematology
Introduction
The peroxidation of lipids and the consequent generation of
bioactive lipid oxidation products are believed to play important
roles in the pathogenesis of atherosclerosis and other inflammatory
processes.1-5 Lipoxygenase, cyclooxygenase, and cytochrome P450
are considered the primary enzymatic participants in these events.6-10
These enzymes are expressed in leukocytes and catalyze the direct
insertion of molecular oxygen (O2) into polyenoic fatty acids,
forming hydroperoxides and other advanced oxidation products.8
Whether alternative chemical pathways contribute to the oxidation
of lipoproteins and lipids in complex biologic matrices, such as
plasma, has not yet been fully defined.
We and others have proposed that another potential pathway for
initiating lipid peroxidation in vivo may involve myeloperoxidase
(MPO), a heme protein present in neutrophils, monocytes, and
certain subpopulations of tissue macrophages.11-13 On phagocyte
activation in peripheral tissues and fluids, MPO is secreted into the
extracellular milieu and into the phagolysosome, where it uses
hydrogen peroxide (H2O2) generated during a respiratory burst as a
cosubstrate. Activated intermediates, Compounds 1 and 2, are
sequentially formed that generate reactive oxidants and diffusible
radical species through 2- and 1-electron oxidation reactions,
respectively.5,14 At plasma levels of halides, chloride (Cl⫺) is a
major cosubstrate for MPO and the cytotoxic oxidant hypochlorous
acid (HOCl) is produced.15 In addition to halides16 and the
pseudohalide thiocyanate (SCN⫺),17,18 various organic and inorganic components found in plasma have been suggested to
potentially serve as naturally occurring substrates for MPO. These
include, but are not limited to, estrogens,19 catecholamines,20
22
23
24
25
tyrosine,21 nitrite (NO⫺
2 ), ascorbate, NADPH, serotonin, and
26
nitric oxide (nitrogen monoxide, NO). The relative contribution
of peroxidation of these substrates to the overall activity of MPO in
vivo is unknown. The products they form and the reactions they
initiate may serve significant biologic functions. For example,
NO⫺
2 , a major end-product of NO metabolism, and tyrosine
undergo MPO-catalyzed 1-electron oxidation reactions generating
nitrogen dioxide ( •NO2)22,27 and tyrosyl radical,21 respectively.
These diffusible oxidant species have been shown to initiate lipid
peroxidation in model systems using isolated lipoproteins or lipid
vesicles as targets.11-13 Moreover, protein oxidation products that
can be formed by MPO-generated tyrosyl radical or •NO2, such as
dityrosine28 and nitrotyrosine,22,29,30 have been observed in human
atherosclerotic lesions31-33 and other inflammatory sites at which
From the Department of Cell Biology, the Department of Cardiology, and the
Center for Cardiovascular Diagnostics, Preventive Cardiology Section,
Cleveland Clinic Foundation, OH; the Chemistry Department, Cleveland State
University, OH; and the Inflammation Program and Department of Medicine,
University of Iowa and Veterans Administration Medical Center, Iowa City.
Association. Z.S. is the recipient of a Jane Coffin Childs Memorial Fund for
Medical Research Fellowship.
Submitted July 12, 2001; accepted October 23, 2001.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by National Institutes of Health grants HL62526 and HL61878
(S.L.H.) and a Merit Review Grant from the Veterans Administration (W.M.N.).
R.Z. is supported by a postdoctoral fellowship from the American Heart
1802
Reprints: Stanley L. Hazen, Cleveland Clinic Foundation, Lerner Research
Institute, Dept of Cell Biology, 9500 Euclid Ave, NC-10, Cleveland, OH 44195;
e-mail: [email protected].
© 2002 by The American Society of Hematology
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
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BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
MYELOPEROXIDASE-MEDIATED OXIDATION OF PLASMA LIPIDS
lipid peroxidation products and MPO are also found.34-36 Although
in vitro studies examining individual potential substrates for MPO
in isolation have suggested their potential role in promoting lipid
oxidation, the actual substrates used by MPO in complex biologic
tissues and fluids in vivo have not yet been determined. Indeed, a
role for MPO in the oxidation of low-density lipoprotein (LDL) and
in the initiation of lipid peroxidation has recently been questioned
by several investigators. Noguchi et al37 examined the capacity of
leukocytes isolated from wild-type and MPO knockout mice to
promote the oxidation of LDL in model systems ex vivo and
observed only modest differences in the parameters of lipid
oxidation monitored.37 It has also recently been suggested that
MPO-catalyzed oxidation of LDL is inhibited, rather than promoted, by the presence of NO⫺
2 , particularly when focusing on
protein oxidation products.38 Moreover, an anti-oxidant rather than
a pro-oxidant function for MPO-generated tyrosine oxidation
products and LDL oxidation has been proposed.39,40 It has also been
suggested by some investigators that HOCl generated by MPO can
promote the oxidation of lipoprotein lipids and the formation of
hydroperoxides,41 whereas other studies have not supported these
observations.11,30 Finally, recent studies have noted species differences between murine and human leukocytes with respect to MPO
and the generation of reactive oxidant species.42-45
In the current study we sought to definitively establish whether
human leukocytes use MPO for catalyzing the oxidation of lipids in
complex biologic matrices, such as in plasma, where numerous
competing cosubstrates for the enzyme are present. We also sought
to identify chemically the component(s) in plasma that serve as
preferred substrates for the MPO–H2O2 system of leukocytes for
the initiation of lipid peroxidation. In studies using leukocytes
isolated from healthy and MPO-deficient subjects, in combination
with HPLC with on-line electrospray ionization tandem mass
spectrometry (LC/ESI/MS/MS), we now show that human neutrophils use MPO to initiate lipid peroxidation in whole plasma
through multiple distinct diffusible substrates.
involving human subjects of either the Cleveland Clinic Foundation or the
University of Iowa and were approved by their respective institutional
review boards. Informed consent was provided according to the Declaration
of Helsinki.
Data are presented as mean ⫾ SD. Comparisons between control and
tested groups were made using nonparametric analysis, and P ⬍ .05 was
considered significant between 2 groups.
Materials and methods
Chemicals and reagents
Hanks balanced salt solution (HBSS) was purchased from Gibco BRL
(Grand Island, NY). Free fatty acids were purchased from Cayman
Chemical (Ann Arbor, MI). Organic solvents were obtained from Fisher
Scientific (Pittsburgh, PA). All other reagents were purchased from Sigma
Chemical (St Louis, MO) unless otherwise indicated.
General procedures
All buffers were passed over a Chelex-100 resin column (Bio-Rad,
Hercules, CA) and were supplemented with diethylenetriamine pentaacetic
acid (DTPA) to remove potential contaminant transition metal ions that
might catalyze LDL oxidation during incubation. Protein content was
determined by the Markwell-modified Lowry protein assay with bovine
serum albumin as standard.46 The concentration of reagent H2O2 was
assayed spectrophotometrically (⑀240 ⫽ 39.4 M⫺1cm⫺1).47 Production of
H2O2 by glucose and glucose oxidase (GO, EC 1.1.3.4) was determined by
the oxidation of Fe(II) and the formation of an Fe(III)–thiocyanate
complex.22 Peroxidase activity in isolated leukocytes was evaluated by
determining the rate of guaiacol oxidation.48 HOCl production was
quantified by the taurine chloramine method.49 Superoxide (O2•⫺) production by activated human neutrophils was measured as the superoxide
dismutase (SOD)–inhibitable reduction of ferricytochrome c.50 In situ
staining of native gels for peroxidase activity was performed as described.51
All protocols were in accordance with institutional guidelines on research
1803
MPO and lipoprotein isolation
MPO (donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7) was isolated and characterized as described.28,51 Purity of isolated MPO was
established by demonstrating R/Z ⱖ 0.85 (A430/A280), sodium dodecyl
sulfate–polyacrylamide gel electrophoresis analysis with Coomassie blue
staining, and in-gel tetramethylbenzidine peroxidase staining to confirm no
eosinophil peroxidase contamination.51 Purified MPO was stored in 50%
glycerol at ⫺20°C. Enzyme concentration was determined spectrophotometrically (⑀430 ⫽ 170 000 M⫺1cm⫺1).52 LDL was isolated from fresh
plasma by sequential ultracentrifugation as a 1.019 ⬍ d ⬍ 1.063 g/mL
fraction, and dialysis was performed in sealed jars under argon atmosphere.53 Final preparations were kept in 50 mM sodium phosphate (pH
7.0), 100 ␮M DTPA, and were stored under N2 until use. LDL concentrations are expressed per milligram LDL protein.
Human neutrophil preparations
Human neutrophils were isolated from whole blood obtained from healthy
and MPO-deficient subjects, as described.54 Neutrophil preparations were
suspended in HBSS (Mg2⫹-, Ca2⫹-, phenol-, and bicarbonate-free, pH 7.0)
and were used immediately for experiments. Molecular characterization of
2 of the MPO-deficient subjects have been reported in previous
publications.55,56
Lipid peroxidation reaction
Isolated human neutrophils (106/mL) were incubated at 37°C with either
50% (vol/vol) normal human plasma or isolated human LDL (0.2 mg/mL)
under air in HBSS supplemented with 100 ␮M DTPA. Neutrophils were
activated by adding 200 nM phorbol myristate acetate (PMA) and were
maintained in suspension by gentle mixing every 5 minutes. After 2 hours,
reactions were stopped by immersion in ice or water bath, centrifugation at
4°C, and immediate addition of 50 ␮M butylated hydroxytoluene (BHT)
and 300 nM catalase to the supernatant. Lipid peroxidation products in the
supernatant were then rapidly assayed as described below.
Reactions with isolated MPO were typically performed at 37°C in
sodium phosphate buffer (20 mM, pH 7.0) supplemented with 100 ␮M
DTPA using 30 nM MPO, 1 mM glucose (G), and 20 ng/mL GO. Under
this condition, a constant flux of H2O2 (0.18 ␮M/min) was generated by
the glucose–glucose oxidase (G/GO) system. Unless otherwise stated,
reactions were terminated by immersion in ice or water bath and the
addition of 50 ␮M BHT and 300 nM catalase to the reaction mixture.
Lipid extraction and sample preparation
Lipids were extracted and prepared for mass spectrometry analysis under
argon or nitrogen atmosphere at all steps. First, hydroperoxides in the
reaction mixture were reduced to their corresponding hydroxides by adding
SnCl2 (1 mM final). A known amount of deuterated internal standard,
12-HETE-d8 (Cayman Chemical), was added to the sample, and plasma
lipids were extracted by adding a mixture of 1 M acetic acid–2-isopropanol–
hexane (2/20/30, vol/vol/vol) at a ratio of 5 mL organic solvent mix:1 mL
plasma. After vortexing and centrifugation, lipids were extracted to the
hexane layer. Plasma was re-extracted by the addition of an equal volume
of hexane, followed by vortexing and centrifugation. Cholesteryl ester
hydroperoxides (CE-H(P)ODEs) were analyzed as their stable SnCl2reduced hydroxide forms by drying of the combined hexane extracts under
N2, reconstituting samples with 200 ␮L 2-isopropanol–acetonitrile–water
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1804
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
ZHANG et al
(44/54/2, vol/vol/vol) and storage at ⫺80°C under argon until analysis. For
the assay of free fatty acids and their oxidation products, total lipids
(phospholipids, cholesterol esters, triglycerides) were dried under N2 and
resuspended in 1.5 mL 2-isopropanol, and fatty acids were released by base
hydrolysis with 1.5 mL 1 M NaOH at 60°C for 30 minutes under argon.
Hydrolyzed samples were acidified to pH 3.0 with 2 M HCl, and fatty acids
were extracted twice with 5 mL hexane. The combined hexane layers were
dried under N2, resuspended in 100 ␮L methanol, and stored under argon at
⫺80°C until analysis by LC/ESI/MS/MS, as described below.
HPLC fractionation of plasma filtrate
To study the role played by low-molecular–weight compounds in plasma as
substrates for MPO in the promotion of lipid peroxidation, whole plasma
from healthy donors was filtered through a 10-kd MWt cutoff filter
(Centriprep YM-10; Millipore, Bedford, MA) by centrifugation. The filtrate
of plasma was used either directly or after fractionation by HPLC.
Reverse-phase HPLC fractionation was performed using a Beckman C-18
column (4.6 ⫻ 250 mm, 5 ␮m OD; Beckman Instruments, Fullerton, CA).
Separation of low-molecular–weight compounds in plasma filtrate (0.5 mL)
was carried out at a 1.0 mL/min flow rate with 100% mobile phase A (water
containing 0.1% acetic acid) over 10 minutes, followed by a linear gradient
generated with 100% mobile phase B (methanol containing 0.1% acetic
acid) over 10 minutes, followed by 100% mobile phase B over 5 minutes.
Effluent was collected as 1-mL fractions, dried under N2, and resuspended
in buffer (0.1 mL) for analysis. Fractionation of plasma filtrate (0.5 mL) by
strong anion exchange HPLC (SAX-HPLC) was performed on a SPHERIS
HPLC column (4.6 ⫻ 250 mm, 5 ␮m SAX; Phase Separations, Norwalk,
CT). The separation of low-molecular–weight compounds in plasma filtrate
was carried out at the flow rate of 0.9 mL/min under isocratic conditions
using 45 mM ammonium acetate buffer (pH 4.0) as mobile phase. Effluent
was collected as 1-mL fractions, dried under N2, and resuspended in buffer
(0.1 mL) for analysis.
Mass spectrometry
LC/ESI/MS/MS was used to quantify free radical-dependent oxidation
products of arachidonic acid 9-H(P)ETE and linoleic acid 9-H(P)ODE.
Immediately before analysis, 1 vol H2O was added to 5 vol methanolsuspended sample, which was then passed through a 0.22-␮m filter
(Millipore). Sample (20 ␮L) was injected onto a Prodigy C-18 column
(1 ⫻ 250 mm, 5 ␮m OD, 100 A; Phenomenex, Rancho Palos Verdes, CA) at
a flow rate of 50 ␮L/min. Separation was performed under isocratic
conditions using 95% methanol in water as the mobile phase. In each
analysis, the entirety of the HPLC column effluent was introduced onto a
Quattro II triple quandrupole MS (Micromass). Analyses were performed
using electrospray ionization in negative-ion mode with multiple reaction
monitoring of parent and characteristic daughter ions specific for the
isomers monitored. Transitions monitored were mass-to-charge ratio (m/z)
2953171 for 9-HODE, m/z 3193151 for 9-HETE, and m/z 3273184 for
12-HETE-d8. N2 was used as the curtain gas in the electrospray interface.
Internal standard 12-HETE-d8 was used to calculate extraction efficiencies
(greater than 80% for all analyses). External calibration curves constructed
with authentic standards were used to quantify 9-HETE and 9-HODE.
Results
Neutrophils isolated from MPO-deficient subjects fail to initiate
lipid peroxidation in plasma but do so after the addition
of isolated MPO
More than a decade ago, Frei et al57 made the seminal observation
that neutrophil activation in plasma results in the time-dependent
formation of lipid hydroperoxides. The precise mechanism involved was not identified but was characterized by its sensitivity to
ascorbate, which had to be depleted before lipid oxidation products
were formed.57 To test the hypothesis that MPO might serve as the
enzymatic catalyst for leukocyte-dependent peroxidation of plasma
lipids, we compared neutrophils from healthy and MPO-deficient
subjects. Lack of functional MPO activity was confirmed by the
absence of HOCl production after leukocyte activation (Table 1)
and the absence of an MPO activity band within native gels of
leukocyte detergent extracts after in-gel tetramethylbenzidine peroxidase staining (data not shown). MPO-deficient neutrophils
displayed enhanced agonist-dependent O2•⫺ generation relative to
comparably treated normal neutrophils (Table 1), as previously
reported.58-60 Neutrophils isolated from healthy and MPO-deficient
subjects failed to generate detectable levels of NO or NO⫺
2 in a
2-hour period.
To determine the role of MPO in promoting lipid oxidation in
plasma exposed to activated neutrophils, we next incubated cells
with whole plasma (50%, vol/vol) and physiological levels of Cl⫺
(100 mM final). Phagocytes were activated with PMA, and the
formation of 9-H(P)ODE and 9-H(P)ETE, specific oxidation
products of linoleic and arachidonic acids, respectively, was
determined by LC/ESI/MS/MS. Normal neutrophils generated
significant levels of 9-H(P)ODE and 9-(H)PETE in plasma after
cell activation by PMA (Figure 1). In stark contrast, MPO-deficient
neutrophils failed to generate significant levels of lipid peroxidation products after stimulation with PMA, despite their enhanced
capacity to produce O2•⫺. Addition of catalytic amounts of MPO
restored the capacity of MPO-deficient neutrophils to initiate the
peroxidation of endogenous plasma lipids (Figure 1).
Characterization and reaction requirements for peroxidation of
endogenous plasma lipids by activated human neutrophils
and isolated human MPO
Addition of catalase, but not heat-inactivated catalase, to cell
mixtures resulted in the near complete ablation of lipid peroxidation in plasma, strongly suggesting a critical role for H2O2 in the
cell-dependent reaction (Figure 2). Incubation of reaction mixtures
Table 1. Characterization of reactive oxidant species generated by neutrophils
isolated from healthy and MPO-deficient subjects
HOCl production
(nmol/106 per mL/h)
O2•⫺ production
(nmol/106 per mL/h)
29 ⫾ 4.1
77 ⫾ 14
0.2 ⫾ 0.2
125 ⫾ 38
Healthy subjects
Reverse-phase HPLC quantification of CE-H(P)ODEs
Samples (100 ␮L) reconstituted in methanol (without base hydrolysis) were
injected onto a Beckman C-18 column (4.6 ⫻ 250 mm, 5 ␮m OD;
Beckman Instruments). Lipids were separated using an isocratic solvent
system composed of 2-isopropanol–acetonitrile–water (44/54/2, vol/vol/
vol) at a flow rate of 1.5 mL/min. CE-H(P)ODEs were quantified as their
stable hydroxide forms by UV detection at 234 nm using CE-9-HODE
(Cayman Chemical) for generation of an external calibration curve.
PMN (MPO⫹)
MPO-deficient subjects
PMN (MPO⫺)
Neutrophils (1 ⫻
isolated from healthy and MPO-deficient patients were
incubated at 37°C in HBSS (pH 7.0) supplemented with 100 ␮M DTPA. Cells were
activated by the addition of phorbol myristate acetate (200 nM), and cellular
production of either HOCl or O2•⫺ was determined as described under “Materials and
methods.” Results represent the mean ⫾ SD of neutrophil preparations isolated from
4 distinct subjects in each group.
106/mL)
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BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
MYELOPEROXIDASE-MEDIATED OXIDATION OF PLASMA LIPIDS
1805
chlorinating intermediates as the primary oxidants for initiation of
lipid peroxidation in plasma. To confirm a physiological role for
MPO, we next added purified human MPO and an H2O2-generating
system (G/GO) to plasma and monitored the formation of specific
oxidation products by LC/ESI/MS/MS analysis. Formation of
9-H(P)ODE and 9-H(P)ETE occurred readily and had an absolute
requirement for the presence of MPO and the H2O2-generating
system (Figure 3). Lipid oxidation was again inhibited by catalase,
azide, or ascorbate but was not affected by the addition of SOD or
methionine (Figure 3). Collectively, these results strongly support a
pivotal role for the MPO–H2O2 system of leukocytes as a primary
mechanism for initiating lipid peroxidation in complex biologic
tissues and fluids such as plasma.
Figure 1. MPO-deficient neutrophils fail to initiate lipid peroxidation in plasma.
Neutrophils (1 ⫻ 106/mL) isolated from healthy and MPO-deficient subjects were
incubated at 37°C in HBSS supplemented with DTPA (100 ␮M, pH 7.0) and fresh
human plasma (50%, vol/vol). Cells were activated by the addition of phorbol
myristate acetate (PMA, 200 nM) and incubated for 2 hours (Complete System). The
contents of 9-H(P)ODE and 9-H(P)ETE formed within endogenous plasma lipids
were then determined by LC/ESI/MS/MS as described in “Materials and methods.”
Where indicated, human MPO (30 nM) was added to reaction mixtures. Data
represent the mean ⫾ SD of triplicate determinations. Each bar within a cluster for a
given condition represents results obtained from independent experiments performed with neutrophil preparations from a distinct donor. PMN(MPO⫹), neutrophils
isolated from healthy subjects; PMN(MPO⫺), neutrophils isolated from MPOdeficient subjects.
with SOD failed to attenuate the oxidation of plasma lipids (Figure
2). In contrast, addition of heme poisons (eg, azide, cyanide) and
water-soluble antioxidant ascorbate resulted in complete inhibition
of neutrophil-dependent peroxidation of plasma lipids. Finally,
addition of HOCl scavengers, such as dithiothreitol and the
thioether methionine, failed to attenuate neutrophil-dependent
peroxidation of endogenous plasma lipids, assessed by quantification of 9-H(P)ODE and 9-H(P)ETE (Figure 2).
Results thus far presented strongly suggest that neutrophils use
the MPO–H2O2 system to generate reactive species distinct from
Figure 2. Characterization of neutrophil-dependent initiation of lipid peroxidation of endogenous plasma lipids. Neutrophils (1 ⫻ 106/mL) isolated from healthy
subjects (PMN) were incubated at 37°C in HBSS supplemented with DTPA (100 ␮M,
pH 7.0) and fresh human plasma (50%, vol/vol). Cells were activated by the addition
of phorbol myristate acetate (200 nM) and then incubated for 2 hours (Complete
System). The contents of 9-H(P)ODE and 9-H(P)ETE formed within endogenous
plasma lipids were then determined by LC/ESI/MS/MS as described in “Materials and
methods.” Additions or deletions to the Complete System were as indicated. Final
concentrations of additions to the Complete System were 30 nM human MPO, 1 mM
NaN3, 300 nM catalase, 300 nM heat-inactivated catalase, 100 ␮M methionine, 100
␮M ascorbate, and 10 ␮g/mL SOD. Data represent the mean ⫾ SD of 3 independent
experiments.
Endogenous low-molecular–weight substances in plasma
serve as cosubstrates for the MPO-catalyzed initiation
of lipid peroxidation in whole plasma
The active site of MPO sits at the base of a deep and narrow heme
pocket inaccessible to compounds significantly larger than a
dipeptide.61 Thus, the ability of isolated MPO and of an H2O2generating system to initiate lipid peroxidation in plasma is
consistent with low-molecular–weight compounds in plasma serving as cosubstrates for MPO to generate diffusible species capable
of conveying oxidizing equivalents from the heme group to distant
targets, such as plasma lipoproteins. To test this hypothesis,
isolated human LDL was incubated with MPO and an H2O2generating system to generate a physiological flux of H2O2. In the
absence of other cosubstrates, no significant oxidation of lipoprotein lipids was observed (Figure 4). In contrast, the addition of
low-molecular–weight constituents recovered from plasma that
had been filtered through a 10-kd MWt cutoff filter reconstituted
the capacity of the MPO–H2O2 system to promote lipid peroxidation (Figure 4, left). In a parallel set of experiments, either plasma
or dialyzed plasma was exposed to the MPO–H2O2 system, and the
Figure 3. Characterization of MPO-dependent initiation of lipid peroxidation of
endogenous plasma lipids. Fresh human plasma (50%, vol/vol) was incubated with
isolated human MPO (30 nM) at 37°C in HBSS supplemented with DTPA (100 ␮M,
pH 7.0) and an H2O2-generating system composed of G/GO for 12 hours (Complete
System). Under this condition, a continuous flux of H2O2 is formed at 10 ␮M/h. The
contents of 9-H(P)ODE and 9-H(P)ETE formed within endogenous plasma lipids
were then determined by LC/ESI/MS/MS as described in “Materials and methods.”
Additions or deletions to the Complete System were as indicated. Final concentrations of additions to the Complete System were 1 mM NaN3, 300 nM catalase, 300 nM
heat-inactivated catalase, 200 nM SOD, 100 ␮M methionine, and 100 ␮M ascorbate.
Data represent the mean ⫾ SD of 3 independent experiments.
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ZHANG et al
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
SCN⫺, and 17␤-estradiol) comigrated with fractions that reconstituted the capacity of MPO–H2O2 systems to initiate lipid peroxidation. Results of fractionation on a strong anion exchange column
are shown in Figure 6. Consistent with prior results using
reverse-phase HPLC (Figure 5), 17␤-estradiol, tyrosine, NO⫺
2 , and
SCN⫺ coeluted with the major fractions that supported MPOdependent initiation of lipid peroxidation (Figure 6).
Confirmation that MPO uses physiologically relevant levels of
nitrite, SCNⴚ, and tyrosine as cosubstrates to promote
peroxidation of lipids in plasma and demonstration that
17␤-estradiol can be used by MPO to initiate LDL lipid
oxidation under certain conditions
Figure 4. MPO uses endogenous low-molecular–weight/dialyzable substrates
in plasma to initiate lipid peroxidation. (left) LDL (0.2 mg/mL) was incubated with
isolated human MPO (30 nM) and an H2O2-generating system composed of G/GO for
12 hours at 37°C in HBSS supplemented with DTPA (100 ␮M, pH 7.0) as in Figure 3.
Where indicated, the filtrate of plasma (Fp), the low-molecular–weight constituents
derived from plasma filtered through a 10-kd MWt cutoff filter, was added (50%,
vol/vol) to the MPO–LDL reaction mixtures. Isolated MPO, an H2O2-generating
system (G/GO) and LDL were added to Fp (Complete System). The content of
9-H(P)ETE formed within endogenous plasma lipids was then determined by
LC/ESI/MS/MS as described in “Materials and methods.” (right) Plasma (P) or
dialyzed plasma (DP) was incubated with isolated MPO and an H2O2-generating
system (G/GO) under conditions similar to those described above. The content of
9-H(P)ETE formed within endogenous plasma lipids was then determined by
LC/ESI/MS/MS as described in “Materials and methods.” Where indicated, DP and P
were added.
Levels of NO⫺
2 in the plasma of healthy human subjects measure
approximately 3.6 to 5 ␮M, but they may reach 50 ␮M at sites of
inflammation.22 The concentration of free tyrosine in plasma
typically varies between 44 ␮M and 72 ␮M in healthy fasting
subjects.62 Each has been previously shown to be used by MPO as
substrate to initiate lipid oxidation in simple model systems,11,13 but
neither has been reported to initiate MPO-dependent oxidation of
endogenous lipids in plasma. To assess a potential role for NO⫺
2
extent of lipid peroxidation was determined. Lipid peroxidation
occurred in plasma, but not in dialyzed plasma, exposed to the
MPO–H2O2 system (Figure 4, right), suggesting that MPO used
low-molecular–weight substrates within plasma to initiate peroxidation of plasma lipids. Consistent with this observation, the
subsequent addition of plasma filtrate to reaction mixtures using
dialyzed plasma as the target for oxidation restored the ability of
the MPO–H2O2 system to promote lipid peroxidation (Figure
4, right).
HPLC fractionation and identification of low-molecular–weight
substrates in plasma used by MPO for initiation
of lipid peroxidation
To identify the component(s) within the filtrate of plasma that
served as physiological cosubstrates for MPO and that promoted
peroxidation of plasma lipids, plasma filtrate was fractionated on a
reverse-phase HPLC column, and the ability of each fraction to
provide substrates for MPO-dependent oxidation of LDL surface
and core lipids was determined (Figure 5). Comparisons with the
retention times for potential candidate substrates for MPO suggested that the early eluting substrate(s) in fraction 3 comigrated
with low-molecular–weight organic anions and SCN⫺, fraction 4
with NO⫺
2 , fraction 8 with tyrosine and fraction 26 with estradiol
(Figure 5). HPLC with on-line electrospray ionization tandem mass
spectrometry analysis (positive ion mode) demonstrated that fraction 8 was composed almost exclusively of an analyte with m/z
182.08—the m/z anticipated for the molecular cation of tyrosine
(data not shown). The high salt content of fractions 3 and 4
prevented analysis by LC/ESI/MS/MS.
To further identify and confirm the cosubstrates of MPO in
plasma that support the initiation of lipid peroxidation by the
enzyme, plasma filtrate was fractionated by alternative column
chromatographies (ion exchange, straight phase). Under every
chromatography system examined, 4 compounds (tyrosine, NO⫺
2,
Figure 5. Reverse-phase HPLC fractionation and identification of low-molecular–
weight components in plasma used by MPO to initiate lipid peroxidation.
Plasma was filtered through a 10-kd MWt cutoff filter. The filtrate of plasma containing
the low-molecular–weight components was then fractionated by reverse-phase
HPLC as described in “Materials and methods.” Each column fraction was dried
under N2, reconstituted in 50 mM sodium phosphate buffer (pH 7.0), and incubated
with a lipid source (LDL, 0.2 mg/mL), isolated human MPO (30 nM), and an
H2O2-generating system (G/GO) (10 ␮M/h flux of H2O2). After incubation at 37°C for
12 hours, the contents of 9-HETE, 9-HODE, and CE-HODEs were then determined
as described in “Materials and methods.” Retention times of some compounds
described as MPO substrates in vitro include: Cl⫺ and Br⫺, fraction 2 (F2); SCN⫺, F3;
NO2⫺, F4; ascorbic acid, F5; tyrosine, F8; 6-hydroxy-dopamine, F19; serotonin, F20;
catecholamines, F18-23; estradiols, F26.
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BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
MYELOPEROXIDASE-MEDIATED OXIDATION OF PLASMA LIPIDS
1807
present at physiological levels, tyrosine and NO⫺
2 augmented
MPO–H2O2-dependent initiation of lipid oxidation under all conditions examined (data not shown). With physiologically relevant
concentrations, SCN⫺, but not 17␤-estradiol, was able to further
promote lipid peroxidation in the presence of multiple competing
cosubstrates for MPO, including Cl⫺, Br⫺, NO⫺
2 , and tyrosine
(Figure 8, right).
Discussion
Figure 6. Strong anion exchange HPLC fractionation and identification of
low-molecular–weight components in plasma used by MPO to initiate lipid
peroxidation. Plasma was filtered through a 10-kd MWt cutoff filter. The filtrate
containing the low-molecular–weight components was then fractionated by HPLC
using a strong anion exchange column as described in “Materials and methods.”
Each fraction was then assessed for its capacity to provide cosubstrate for the
MPO–H2O2 system and initiate LDL lipid peroxidation as in Figure 5. Retention times
of some compounds described as MPO substrates in vitro include: serotonin,
estradiols, Cl⫺, fraction 3 (F3); tyrosine, F4; ascorbic acid, F5; NO2⫺, F6; SCN⫺, F7.
and tyrosine as cosubstrates involved in lipid oxidation by the
MPO–H2O2 system, we incubated physiologically relevant levels
of each with dialyzed plasma as a lipid source (50%, vol/vol) and
plasma levels of Cl⫺ (100 mM). As shown in Figure 7, even in the
presence of the competing cosubstrate Cl⫺, biologically relevant
concentrations of NO⫺
2 and tyrosine were effectively used by the
MPO–H2O2 system to promote peroxidation of endogenous plasma
lipids. Similar dose-dependent effects of NO⫺
2 and tyrosine as
substrates were noted for MPO-mediated peroxidation of core LDL
lipids in buffer containing plasma levels of halides (Figure 8, left).
Addition of both NO⫺
2 and tyrosine resulted in lipid peroxidation,
but at a level lower than that observed with either substrate alone
(not shown).
Although studies have shown that SCN⫺ and 17␤-estradiol may
serve as reducing substrates for peroxidases in vitro, the capacity of
MPO to use either compound for initiating lipid oxidation has not
yet been reported. SCN⫺ levels in plasma from a healthy subject
range from 17 to 69 ␮M and may reach more than 200 ␮M in the
plasma of smokers.62 Normal levels of total estrogens in serum are
reported to be 37 to 184 pM in men and 18 to 1266 pM in women,
and they can reach significantly higher levels with supplementation
and pregnancy.62 When 17␤-estradiol or SCN⫺ was incubated with
MPO, an H2O2-generating system and LDL in media containing
plasma levels of Cl⫺ lipid peroxidation was initiated in a dosedependent fashion (Figure 8, left). When all cosubstrates were
MPO is the single most abundant protein in neutrophils, but the
precise substrates used and the biochemical reactions mediated by
this enzyme in vivo are still not fully defined. MPO uses Cl⫺ in
vivo to generate microbicidal and chlorinating oxidants, as confirmed by the detection of chlorinated products at sites of inflammation in mice45 and humans.63 Furthermore, selective defects in host
defenses (eg, against fungal and yeast pathogens) are noted in mice
and humans deficient in MPO activity.45,64,65 However, a complex
array of potential reactions in addition to those involving halogenating oxidants have been identified for MPO in model systems.14-30
Numerous low-molecular–weight organic and inorganic substances found in biologic matrices may serve as cosubstrates for
MPO catalysis in isolation in vitro, generating reactive oxidants
and diffusible radical species (reviewed in Podrez et al5 and Kettle
and Winterbourn14). The relative importance of these species and
their contributions to reactions that occur in vivo is uncertain.
Although lipid peroxidation and lipid-derived signaling molecule formation are believed to be critical in atherosclerosis and
other inflammatory disorders, the pathways responsible for these
processes in vivo are not fully established. Leukocyte activation in
whole plasma has long been appreciated as a physiological
mechanism for promoting peroxidation of endogenous plasma
lipids.57 However, the enzymatic participant(s) and the reactive
intermediates involved in leukocyte-mediated lipid oxidation within
complex matrices such as plasma have not been directly defined.
Results of the current study definitively identify MPO as a major
enzymatic catalyst for promoting lipid oxidation by activated
human neutrophils in plasma. Furthermore, the MPO–H2O2 system
uses low-molecular–weight components in plasma distinct from
Cl⫺ for initiating lipid peroxidation at sites of inflammation,
⫺
including tyrosine, NO⫺
2 , SCN , and perhaps 17␤-estradiol.
Figure 7. MPO uses NO2ⴚ and tyrosine as substrates to promote peroxidation of
endogenous plasma lipids under physiologically relevant conditions. Isolated
MPO (30 nM) and an H2O2-generating system (G/GO) (10 ␮M/h flux of H2O2) were
incubated with dialyzed plasma (50%, vol/vol) and the indicated concentrations of
NO2⫺ and tyrosine in 50 mM sodium phosphate buffer (pH 7.0), supplemented with
100 ␮M DTPA and 100 mM NaCl. After incubation at 37°C for 12 hours, the contents
of 9-H(P)ODE and 9-H(P)ETE were then determined as described in “Materials and
methods.”
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1808
ZHANG et al
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
Figure 8. MPO-initiated oxidation of LDL lipids in the
presence of multiple competing cosubstrates. (left)
Isolated MPO (30 nM) and an H2O2-generating system
(G/GO) (10 ␮M/h flux of H2O2) were incubated with LDL
(0.2 mg/mL) and the indicated concentrations of either
SCN⫺, 17␤-estradiol, NO2⫺, or tyrosine in 50 mM sodium
phosphate buffer (pH 7.0), supplemented with 100 ␮M
DTPA and 100 mM NaCl. After incubation at 37°C for 12
hours, the content of CE-H(P)ODE was then determined
as described in “Materials and methods.” (right) Isolated
MPO (30 nM), an H2O2-generating system (G/GO) (10
␮M/h flux of H2O2), and physiological levels of Cl⫺ (100
mM), Br⫺ (100 ␮M), NO2⫺ (5 ␮M), tyrosine (100 ␮M), and
LDL (0.2 mg/mL) were collectively incubated with the
indicated concentrations of SCN⫺ and 17␤-estradiol in 50
mM sodium phosphate buffer (pH 7.0), supplemented
with 100 ␮M DTPA. After incubation at 37°C for 12 hours,
the content of CE-H(P)ODE was then determined as
described in “Materials and methods.”
Recent studies using leukocytes isolated from wild-type and
MPO knockout mice report modest differences in their capacity to
promote lipoprotein lipid oxidation and inhibition in lipid oxidation
by the addition of SOD.37 Two major species differences between
murine and human neutrophils include the significant generation of
NO by mouse but not human neutrophils and the 6- to 10-fold
decrease in MPO content found in murine neutrophils compared
with that observed in humans.30,42,43 Moreover, mixed leukocyte
preparations rather than isolated neutrophils were used in the
studies with MPO knockout mice.37 Whether these factors contributed to the limited role reported for MPO by the elicited murine
leukocytes and the sensitivity to SOD remains to be established.
Neutrophils from multiple unrelated MPO-deficient humans all
failed to promote peroxidation of endogenous lipids in plasma
unless supplemented with catalytic levels of purified MPO. Catalase, peroxidase inhibitors, and ascorbate, but not SOD, inhibited
leukocyte-dependent peroxidation of plasma lipids, consistent with
the MPO–H2O2 system as the responsible mechanism. The current
results thus strongly support a major physiological role for MPO in
initiating lipid peroxidation by activated human leukocytes and
suggest that a function of the enzyme at sites of inflammation may
be to generate lipid oxidation products with biologic activity.
Four compounds in human plasma could support MPOdependent peroxidation of LDL lipids in the presence of physiolog⫺
ical levels of Cl⫺: NO⫺
2 , tyrosine, SCN , and 17␤-estradiol.
⫺
Tyrosine and NO2 have been implicated in the modification of
lipoproteins by the MPO system of leukocytes in prior studies.11-13
However, as far as we are aware, a role for either SCN⫺ or
17␤-estradiol in the initiation of lipid oxidation by peroxidases has
not yet been reported. The addition of tyrosine and NO⫺
2 to the
MPO–H2O2 system modestly inhibited MPO-dependent lipid oxidation compared with that observed with either NO⫺
2 or tyrosine
alone, potentially reflecting the loss of intermediates ( •NO2 and
tyrosyl radical) that promote lipid peroxidation through radical–
radical coupling reactions, generating nitrotyrosine.66
Cigarette smoking is a risk factor for cardiovascular disease,
and plasma levels of SCN⫺ in smokers may be increased 2 to 3
times more than that observed in nonsmokers.67,68 A positive
correlation between serum SCN⫺ levels and the formation of
advanced atherosclerotic plaques within coronary arteries has been
noted.67,69 The current discovery of SCN⫺ as a physiological
reducing substrate for MPO that promotes LDL lipid oxidation
suggests a potential mechanism for this correlation. In recent
studies, hypothiocyanate was identified as a proximate oxidant that
accumulates in buffer during oxidation of SCN⫺ by eosinophil
peroxidase, a related leukocyte peroxidase.70 It is unclear how this
2 e⫺ oxidation product of SCN⫺ would initiate lipid oxidation.
Indeed, the current studies suggest that MPO also generates the
1-electron oxidation product, thiocyanyl radical (•SCN), or that the
addition of SCN⫺ to MPO–H2O2 systems promotes the formation
of a protein radical species on MPO that is accessible to lipid
targets. It is interesting that van Dalen and Kettle71 recently
demonstrated the formation of a radical species after MPOcatalyzed oxidation of SCN⫺. Further examination of the mechanisms of how SCN⫺ oxidation by MPO leads to the initiation of
lipid oxidation are warranted because it is tempting to speculate
that this pathway potentially contributes to the enhanced risk for
cardiovascular disease noted in smokers.
Identification of 17␤-estradiol as a potential substrate for MPO
in plasma that can initiate lipid oxidation was unanticipated. The
current studies suggest, however, that 17␤-estradiol does not likely
play a significant role in lipid oxidation mediated by MPO in vivo
unless localized consumption of alternative competing cosubstrates, such as tyrosine, SCN⫺, and NO⫺
2 has occurred, and
supraphysiological levels are achieved, such as through supplementation. It is nonetheless interesting that elevated levels of 17␤estradiol in serum have been reported in subjects with acute
myocardial infarction and septic shock.72,73 Although many studies
have suggested that estrogens may serve as antioxidant cardioprotectants,74 recent studies involving more than 3000 women demonstrated that estrogen replacement did not reduce, but possibly
enhanced, the overall rate of coronary events in women with
established coronary artery disease.74-76
Taken together, our results highlight the probable contribution
of MPO in promoting lipid oxidation at sites of inflammation. The
development of peroxidase inhibitors as novel anti-inflammatory
agents thus merits consideration. The sensitivity of leukocyte- and
MPO-dependent oxidation of plasma lipids to ascorbate also has
implications for the choice of anti-oxidant regimen one considers.
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BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
MYELOPEROXIDASE-MEDIATED OXIDATION OF PLASMA LIPIDS
In studies using whole plasma, small unilamellar vesicles, or LDL
as targets, ascorbate inhibits MPO-mediated peroxidation of lipids
far more effectively than that observed with ␣-tocopherol (R.Z. and
S.L.H., unpublished results, January 2000). Finally, the capacity of
the MPO–H2O2 system of leukocytes to form bioactive lipid
oxidation products in plasma suggests that MPO plays a broad role
in inflammation biology and host defenses. Indeed, we recently
demonstrated that exposure of LDL to the MPO–H2O2 system of
activated monocytes in media containing NO⫺
2 converts the lipoprotein to a high uptake form capable of promoting cholesterol
accumulation and foam cell formation.12 Oxidized lipid(s) were
shown to serve as major constituent(s) that facilitated recognition
of the modified lipoprotein by the macrophage scavenger receptor
CD36.12,77 This receptor has recently been implicated in foam cell
formation and atherosclerosis.77,78 Thus, MPO-dependent peroxidation of cell membranes and lipoproteins may serve as a physiological mechanism to promote the uptake of modified and senescent
cellular constituents by CD36⫹ cells.
Results of the first large study evaluating nearly 100 MPOdeficient subjects compared with a control population were recently reported.79 In addition to a modestly increased frequency of
infectious diseases, MPO deficiency was associated with a decreased incidence of cardiovascular events.79 We have demonstrated a strong positive correlation between the level of MPO per
leukocyte and the risk for atherosclerosis in subjects with angiographically defined coronary artery disease status.80 The current
results provide further evidence for a potential mechanism contributing to the many links between MPO and cardiovascular disease.
1809
Acknowledgments
We thank Dave Schmitt for technical assistance. Mass spectrometry experiments were performed at the Cleveland Clinic Foundation Mass Spectrometry Resource within the Center for Cardiovascular Diagnostics and Prevention.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2002 99: 1802-1810
doi:10.1182/blood.V99.5.1802
Defects in leukocyte-mediated initiation of lipid peroxidation in plasma as
studied in myeloperoxidase-deficient subjects: systematic identification of
multiple endogenous diffusible substrates for myeloperoxidase in plasma
Renliang Zhang, Zhongzhou Shen, William M. Nauseef and Stanley L. Hazen
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