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(The FASEB Journal. 2003;17:2209-2220.)
© 2003 FASEB

Isolevuglandins, a novel class of isoprostenoid derivatives, function as integrated sensors of oxidant stress and are generated by myeloperoxidase in vivo

EUGENIA POLIAKOV, MARIE-LUISE BRENNAN*,{ddagger}, JENNIFER MACPHERSON*, RENLIANG ZHANG*,{ddagger}, WEI SHA, LAURA NARINE*, ROBERT G. SALOMON and STANLEY L. HAZEN*,{dagger},{ddagger},1

Department of Chemistry, Case Western Reserve University, Departments of
* Cell Biology and
{dagger} Cardiovascular Medicine, Cleveland Clinic Foundation, and
{ddagger} Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Cleveland, Ohio, USA

1 Correspondence: Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave, NC-10, Cleveland, OH 44195, USA. E-mail: hazens{at}ccf.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolevuglandins (isoLGs) are a family of reactive {gamma}-ketoaldehydes generated by free radical oxidation of arachidonate-containing lipids through the isoprostane pathway. Elevated plasma levels of isoLG protein adducts are observed in subjects with atherosclerosis compared with age/gender-matched controls. However, mechanisms for the generation of isoLGs in vivo are not established. Here we show that free radical-induced peroxidation promoted by the myeloperoxidase (MPO)/H2O2 system of leukocytes serves as one mechanism for the generation of isoLGs in vivo. Using a Candida sepsis model of inflammation, we demonstrate 3.5- and 2.7-fold increases in iso[4]LGE2 and isoLGE2 adducts of plasma proteins after pathogen exposure in wild-type mice. Plasma levels of F2 isoprostanes were not significantly increased after pathogen challenge in this model. MPO knockout mice demonstrated significant reductions (34%, P=0.003) in plasma levels of iso[4]LGE2 protein adducts after pathogen challenge compared with wild-type mice. Mass spectrometry and immunochemical methods demonstrate MPO-dependent formation of iso[4]LGE2 and isoLGE2 phospholipids and their corresponding isoLG protein adducts in model systems. The present studies thus identify MPO as one pathway for generation of isoLGs in vivo. They also suggest that long-lived protein isoLG adducts may serve as an alternative integrated sensor of oxidant stress in vivo.—Poliakov, E., Brennan, M.-L., MacPherson, J., Zhang, R., Sha, W., Narine, L., Salomon, R. G., Hazen, S. L. Isolevuglandins, a novel class of isoprostenoid derivatives, function as integrated sensors of oxidant stress and are generated by myeloperoxidase in vivo.


Key Words: isoprostane • myeloperoxidase • isolevuglandin • oxidant stress


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OXIDANT STRESS and lipid peroxidation are believed to play important roles in the development of inflammatory and vascular diseases (1 2 3 4) . These processes lead to the production of biologically active oxidized lipids (5 6 7 8 9) and to the conversion of low density lipoprotein (LDL) into an atherogenic form (10 11 12) . A variety of methods are used to assess oxidation status in vivo. However, classical methods such as thiobarbituric acid reactive substances (TBARs) and the conjugated diene assay suffer from lack of specificity and limited application to clinical specimens (13) . Measurement of peroxidation products of free polyunsaturated fatty acids (PUFAs) such as malondialdehyde, 4-hydroxy-2-nonenal (HNE), or lipid hydroperoxides is limited by the relative instability of the analytes and their ready formation ex vivo (14) . Measurement of isoprostanes is widely used as a viable alternative for monitoring oxidant stress in vivo (15 16 17 18) . Isoprostanes, products of free radical-induced oxidation of arachidonic acid (AA), are isomers of enzymatically formed prostaglandins. They are relatively stable chemically and can be measured in biological tissue and fluids with good sensitivity and specificity (14 15 16 17 18) . However, their half-life in blood is limited by their rapid metabolism and excretion (14 15 16 , 19 20 21 22 23) . Thus, isoprostanes provide a snapshot of oxidant stress in vivo over a relatively brief period of time but their utility in providing an integrated assessment of oxidant stress over a longer interval may be limited.

Besides isoprostanes, free radical peroxidation of AA produces another class of compounds—isolevuglandins (isoLGs)—through common isoprostanoid endoperoxide precursors (24 , 25) . Isoprostanes include structural and stereoisomers of prostaglandins that may be generated by cyclooxygenase-promoted oxygenation of AA, as well as via free radical-mediated mechanisms. Similarly, isolevuglandins comprise structural isomers (e.g., iso[4]LGE2) as well as stereoisomers (e.g., isoLGE2) of cyclooxygenase-generated levuglandins (e.g., LGE2) and may also be formed via free radical-mediated pathways (24 , 26) . IsoLGs differ from isoprostanes by containing a characteristic aldehydic group in a 1,4-dicarbonyl array, making them extremely reactive toward primary amino groups in proteins (Scheme 1 ). IsoLGs initially form Schiff base adducts, then pyrrole adducts, with the {varepsilon}-amino group of lysyl residues (27 , 28 ). However, the pyrrole adducts are unstable in the presence of oxygen and are further transformed to lactam and hydroxylactam adducts, which accumulate as stable end products (Scheme 1) (29) . Formation of isoLG protein adducts from free AA in vitro has been confirmed immunologically and by a variety of mass spectrometry methods (24 , 25) . Elevated levels of isoLG protein adducts in plasma from patients with atherosclerosis compared with healthy age-matched subjects have been shown using polyclonal antibodies raised against synthetic isoLG-pyrrole-derived adducts (30) . Collectively, these data suggest that isoLGs and their protein adducts may serve as markers of oxidant stress. However, mechanisms for generation of isoLGs in vivo have not been established.



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  		Scheme 1. Structure of isoLGE2 and its protein adducts.

Recent studies reveal that myeloperoxidase (MPO) serves as an enzymatic catalyst for initiation of lipid peroxidation and lipoprotein oxidation in vivo (31) . MPO is an abundant heme protein secreted by phagocytes in response to stimulation (32) . MPO (33) and its distinct products [HOCl-damaged proteins (34) and 3-chlorotyrosine (35) ] are enriched in human atherosclerotic aortic intima and LDL recovered from atheroma. MPO uses H2O2 together with low molecular weight cosubstrates like chloride (36) , tyrosine (37) , and nitrite (NO2-) (38 , 39) to generate a variety of reactive oxidants and diffusible radical species (3 , 40) . Recent studies using MPO knockout mice reveal that NO2-, the autoxidation product of nitric oxide, serves as a preferred substrate for MPO to generate nitrogen dioxide, a species capable of aromatic nitration and initiation of lipid peroxidation in vivo (31 , 39) . MPO knockout mice are more susceptible to Candida infection, making the Candida sepsis model a useful tool for studying the role of MPO in inflammation (41 42 43) .

We now report significant increases in isoLG protein adducts in plasma proteins of mice after Candida sepsis using enzyme-linked immunosorbent assays (ELISA) with antibodies specific for isoLG protein adducts. Plasma levels of F2 isoprostanes failed to significantly increase in wild-type or MPO knockout mice in this model. Comparison of plasma levels of iso[4]LGE2 protein adducts in wild-type vs. MPO knockout mice revealed a significant reduction in levels within plasma recovered from MPO knockout mice. Parallel in vitro studies using reverse phase HPLC with on-line electrospray ionization tandem mass spectrometry and immunochemical methods confirmed the ability of the MPO-H2O2-NO2- system to form isoLGs and isoLG protein adducts from target phospholipid vesicles and lipoproteins, respectively. The present findings thus confirm that MPO serves as one pathway for generation of isoLGs in vivo. They also suggest that monitoring longer lived protein/lipid adducts in plasma may be a more sensitive means than F2 isoprostanes of detecting enhanced oxidant stress in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
All solvents were purchased from Fisher Scientific Inc. (HPLC grade). Alkaline phosphatase-coupled goat anti-rabbit immunoglobulin (IgG), catalase (bovine liver, thymol-free), and glucose oxidase (grade II) were purchased from Boehringer Mannheim (Indianapolis, IN, USA). LGE2 human serum albumin (HSA), LGE2 bovine serum albumin (BSA), iso[4]LGE2-BSA, iso[4]LGE2-HSA, 4-oxononanal (ON) coupled BSA (ON-BSA), and ON-HSA were prepared as described previously (24 , 44 , 45) . ON-coupled keyhole limpet hemocyanin (KLH), iso[4]LGE2-KLH, and LGE2-KLH polyclonal rabbit antibodies were raised against LGE2, iso[4]LGE2, and 4-oxononanal adducts of KLH, as described (24 , 44 , 45) . Protease inhibitor cocktail for general use (P2714) and all other chemicals were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise.

General methods
Absorbance values were measured on a Bio-Rad Microplate Reader using wavelength 405 nm. Protein measurements were done by a modified Lowry method (P5656, kit for protein determination; Sigma). Production of H2O2 by glucose/glucose oxidase (G-GOx) was quantified by oxidation of Fe(II) and formation of a Fe(III)/thiocyanate complex (10) . Total plasma levels of F2 isoprostanes were determined using HPLC with on-line electrospray ionization tandem mass spectrometry and stable isotope dilution methodology, as described previously (31) . All data are presented as mean ± SD unless stated otherwise. Data shown represent results from a representative experiment performed multiple independent times on separate days, as described in the figure legends. Statistical difference is estimated by independent two-tailed Student's t test with P < 0.05 considered significant.

LGE2-bismethoxime and iso[4]-LGE2-bismethoxime synthesis
Iso-LGE2 and iso[4]LGE2 were synthesized from a 15-R,S epimeric mixture of the methyl(or ethyl) ester, O-tert-butyl-dimethylsilyl ether, isopropylidine precursor, as described (24) . Bismethoxime derivatives were synthesized by incubation with methoxylamine hydrochloride in anhydrous pyridine at room temperature for 24 h under inert atmosphere (26) . Products were extracted with ethyl acetate and purified by flash chromatography using 50% ethyl acetate/hexane.

Lipid vesicle preparation, oxidation, and derivatization
Small unilamellar vesicles comprised of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) (2 mg/mL, 1 mL) were prepared by extrusion (10 times) through a 0.1 µm polycarbonate filter (Avanti Mini-extruder set, Avanti Polar Lipids, Inc., Alabaster, AL, USA) in argon-sparged sodium phosphate buffer (50 mM, pH 7.4) supplemented with 200 µM DTPA, as described (6) . Vesicles were then diluted to a final concentration of 0.2 mg of lipid/mL and oxidized by incubation at 37°C for 7 h in the presence of 30 nM MPO, 100 µg/mL glucose, 100 ng/mL glucose oxidase, and 50 µM NaNO2 (10) . Control studies demonstrated that under these conditions a constant flux of H2O2 (0.80 µM/min) was generated by the glucose/glucose oxidase system. Oxidation was stopped by addition of methoxylaminehydrochloride (30% aqueous solution, v/v) to a final concentration of 3% methoxylamine-HCl. Samples were incubated for 45 min at room temperature, treated with an equal volume of a 15% KOH in methanol solution for 30 min at 37°C, then neutralized with 1 N HCl to pH 4.

Fatty acid extraction
Derivatized fatty acids were extracted from phosphate buffer (1 mL) by addition of NaCl (20 mg), followed by 2.5 mL of a solution comprised of isopropanol: heptane: 2M acetic acid (40:10:1, v/v/v). The mixture was vortexed, then heptane (2.5 mL) was added and the mixture was again vortexed. Samples were centrifuged at 4°C for 5–10 min at 3000 rpm and the top (heptane) layer was collected. The bottom layer was reextracted with heptane (2.5 mL), then a third time with hexane (2.5 mL). All heptane and hexane layers were combined and evaporated under nitrogen. The residue was resuspended in 0.5 mL of methanol (HPLC grade) and stored in an amber vial under argon atmosphere at -20°C until analysis.

LDL oxidation by the MPO/G-GOx/NO2- system and Cu2+
LDL was isolated from fresh human plasma by sequential ultracentrifugation as a fraction with density between 1.019 and 1.063 g/mL (10) . LDL (0.2 mg/mL) was typically oxidized at 37°C in sodium phosphate buffer (50 mM, pH 7.0) supplemented with 200 µM DTPA using 30 nM MPO, 100 µg/mL glucose, 20 ng/mL glucose oxidase, and 50 µM NaNO2 for the period indicated. Control studies demonstrated that under these conditions a constant flux of H2O2 (10 µM/h) is generated. Reactions were terminated by addition of 40 µM butylated hydroxytoluene (BHT; from 20 mM ethanolic stock) and 300 nM catalase to the reaction mixture. LDL oxidation by CuSO4 (16 µM) was performed at 37°C in sodium phosphate buffer (50 mM, pH 7.4) for the periods indicated. Reactions were terminated by addition of 40 µM BHT (from 20 mM ethanolic stock) and DTPA (100 µM, pH 7.0) to the reaction mixture.

Candida sepsis model
Wild-type (C57B46J) and MPO knockout mice used were >10 generations backcrossed onto C57B46J background. As a model of inflammation, mice were injected intraperitoneally with 6 x 108 C. albicans. At the indicated times after infarction, mice were bled via retro-orbital sinus into blood collector tubes containing EDTA (2 mM). Plasma was rapidly isolated and BHT (100 µM), pepstain A (5 µM), and a protease inhibitor cocktail (1:250) were added. Samples were overlaid with argon, flash frozen in liquid nitrogen, and kept at -80°C until analysis. Parallel studies characterizing the animal model were also performed. As has been reported in this model (39) , control studies confirmed that wild-type and MPO knockout mice demonstrated comparable cell count, and differentials from recovered peritoneal lavage fluid, white blood cell counts and differentials monitored from whole blood, and comparable levels of alternative enzymatic participants for initiation of lipid peroxidation (12 lipoxygenase, and COX 1, 2) as monitored by Western analysis of isolated leukocytes recovered from peritoneal lavage (data not shown).

Analysis of isolevuglandins
LC/ESI/MS/MS analysis
Mass spectrometric analyses were performed on a Quatro II triple-quadruple mass spectrometer (Micromass, Inc., Beverly, MA, USA) interfaced with an HP 1100 HPLC (Hewlett-Packard, Palo Alto, CA, USA). Derivatized isolevuglandins were resolved on an Ultrasphere ODS C18 column (2x150 mm, 5 µm, Beckman Instruments, Fullerton, CA, USA) equilibrated and run under isocratic conditions at 0.2 mL/min using acetonitrile/water (90:10, v/v) with 0.1% formic acid as the solvent. HPLC with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) analyses of isoLG methoxime derivatives were performed in the positive ion mode with multiple reaction monitoring (MRM) by showing co-chromatography of multiple characteristic parent -> daughter ion transitions for iso-LGE2 [mass to charge ratio (m/z) 393->361, 393->316, 393->238] and for iso[4]LGE2 (m/z 393->361, 393->316, 393->198).

ELISA binding inhibition studies
Competitive ELISAs for LDL and plasma samples were performed as described previously (44 45 46 47) . Mouse plasma was diluted 1:10 prior to assay. Polyclonal rabbit antibodies raised against iso[4]LGE2-, isoLGE2-, and 4-ON-derived protein-bound adducts were used to measure iso[4]LGE2-, isoLGE2, and HNE protein adducts, respectively. Polyclonal antibodies were purified on protein G columns to give IgG fractions as described previously (24 , 44 , 45) . All immunoreactivity measurements in LDL or plasma are expressed per milligram of protein.

In control studies designed to assess the reproducibility of immunochemical and mass spectrometric assays used, measurements of a test set of samples were performed repeatedly on the same day as well as on different days. Coefficients of variance for replicates of analytes monitored within a given assay (same day) were <= 4%. Interassay variability of samples analyzed on multiple independent days for all analytes were <= 7%.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Myeloperoxidase generates isolevuglandins from arachidonate-containing phospholipids in a model system
IsoLG protein adducts are elevated in patients with coronary artery disease (30) , and recent studies show that MPO serves as one pathway for initiation of lipid peroxidation in vivo (31) . Based on these observations, we hypothesized that MPO might serve as a mechanism for generating isoLGs in vivo. As a first step, we performed studies aimed at testing whether or not MPO could generate isoLGs in vitro using well-defined model systems. IsoLGs are extremely labile in biological matrices and not readily detected free in solution. We first performed mass spectrometry studies of synthetic iso[4]LGE2 (Fig. 1 A, B) and isoLGE2 (Fig. 2 A, B) to determine how best to monitor for these species in reaction mixtures. Synthetic iso[4]LGE2 and isoLGE2 were derivatized to their stable methoxime derivatives and their collisionally induced positive ion tandem mass spectra determined (Fig. 1B and Fig. 2B ). Both iso[4]LGE2 and isoLGE2 possess molecular cations ([M+H]+) with mass-to-charge ratio (m/z) 411 and undergo fragmentation patterns with numerous common daughter ions, including generation of a pseudo-parent cation at m/z 393 due to loss of water (MH-H2O)+, m/z 361 (MH-OCH3)+, m/z 331 (MH-2OCH3)+, and m/z 316 (MH-2OCH3-CH3)+ (Fig. 1B and Fig. 2B ). An abundant daughter ion at m/z 198 was unique for the bismethoxime derivative of iso[4]LGE2 (Fig. 1A ), while a daughter ion at m/z 238 was unique for the bismethoxime derivative of isoLGE2 (Fig. 2A ). Formation of these unique daughter ions may be explained by loss of methoxy (OCH3), methyl (CH3) and the lower portion of each class of isoLGE2 molecule (proposed fragmentation pattern shown in inserts, Fig. 1A and Fig. 2A ). Both syn and anti isomers of the oxime derivatives were generated for each isoLGE2 class; in the case of iso[4]LGE2, they were resolvable (Fig. 1A, C , bottom traces) and demonstrated identical fragmentation patterns (not shown). Based on the above results, we developed HPLC with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) methods to individually monitor each distinct isoLG form. Iso[4]LGE2 (Fig. 1C , bottom traces) and isoLGE2 (Fig. 2C , bottom traces) were detected by demonstrating co-chromatography of the transitions between two common and one unique daughter ion of the pseudo-parent ion at m/z 393 (i.e., m/z 393->361, 393->316, and 393->198 for iso[4]LGE2; and m/z 393->361, 393->316, and 393->238 for isoLGE2) using multiple reaction monitoring mode.



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  		Figure 1. Mass spectrometric characterization of iso[4]LGE2 produced in PAPC vesicles exposed to the myeloperoxidase-H2O2-NO2- system. A) LC/MS/MS analysis with MRM (m/z 393 to m/z 361) and structure of the iso[4]LGE2-methoxime standard, B) Positive ion electrospray tandem MS/MS fragmentation pattern for the pseudo-parent ion (m/z 393, [MH-H20]+) of the iso[4]LGE2-methoxime standard. C) Analysis of sample (oxPAPC vesicles) and standard by positive ion tandem LC/ESI/MS/MS with MRM; monitoring transitions as indicated (m/z 393 to m/z 198, m/z 316, and m/z 361).



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  		Figure 2. Mass spectrometric characterization of isoLGE2 produced in PAPC vesicles exposed to the myeloperoxidase-H2O2-NO2- system. A) LC/ESI/MS/MS analyses using MRM (m/z 393 to m/z 361) and structure of the iso-LGE2-methoxime standard. B) Positive ion electrospray tandem MS/MS fragmentation pattern for the pseudo-parent ion (m/z 393, [MH-H20]+) of the iso-LGE2-methoxime standard. C) Analysis of sample (oxPAPC vesicles) and standard by positive ion tandem LC/ESIMS/MS with MRM; monitoring transitions as indicated (m/z 393 to m/z 238, m/z 316, and m/z 361).

Having developed the above LC/ESI/MS/MS methods, we sought to determine whether MPO could generate isoLGE2 species in vitro. Small unilamellar vesicles comprised of PAPC were incubated with isolated human MPO, NO2- and a continuous flux of H2O2 generated by the G/GOx system (see Materials and Methods). Any labile isoLGEs formed within the oxidized phospholipid vesicles were derivatized to their corresponding stable methoxime adducts and monitored by LC/ESI/MS/MS after release of free acid isoLGE2-methoxime derivatives by base hydrolysis. As shown in Fig. 1C and Fig. 2C (upper tracings), MPO generated both isoLGE2 and iso[4]LGE2 species in target PAPC vesicles as each demonstrated comigration of the appropriate transitions between parent and daughter ions, which also comigrated with the methoxime derivatives of authentic standards (Fig. 1C and Fig. 2C ). Further corroboration of isoLGE2 and iso[4]LGE2 formation was detection of both syn and anti isomers of the oxime derivatives of each; for the iso[4]LGE2 species, both syn and anti isomers were resolvable (Fig. 1C ). Collectively, these results confirm that the MPO-H2O2-NO2- system of leukocytes generates isolevuglandins from target bilayers containing esterified arachidonic acid.

The myeloperoxidase oxidation system produces isoLG protein adducts in target lipoproteins
The 1,4 dicarbonyl array of isoLGs renders them chemically reactive with nucleophilic moieties in biological matrices. In the presence of {varepsilon}-amino groups of protein lysyl residues, they readily form pyrrole adducts (29) . MPO-induced oxidation of arachidonyl-containing phospholipids in cell membranes or lipoproteins are thus best detected as isoLG protein adducts. We next examined whether LDL exposed to the MPO/G-GOx system in the presence vs. absence of NO2- produced isoLGE2 adducts. As an independent index of lipid peroxidation-induced protein/lipid adduct formation, we also measured formation of HNE-derived pyrrole adducts in LDL oxidized by the MPO-H2O2-NO2- system. Exposure of LDL to the MPO-H2O2-NO2- oxidation system resulted in time-dependent formation of apolipoprotein B-100 adducts of iso-LGE2, iso[4]LGE2, and HNE (Fig. 3 ). MPO-dependent formation of the lipid protein adducts required the presence of NO2-, consistent with MPO-catalyzed formation of nitrogen dioxide as the diffusible oxidant that initiated lipid peroxidation (31 , 39) .



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  		Figure 3. Time course of formation of (A) isoLGE2, (B) iso[4]LGE2, and (C) HNE protein adducts during LDL oxidation by MPO oxidation system. LDL was incubated at 37°C with the MPO/GGOx system in the presence ({blacksquare}) or absence (•) of NO2- as described in Materials and Methods. Levels of isoLGE2 and HNE protein adducts were determined by ELISA with polyclonal antibodies raised against isoLG- and HNE-derived protein-bound adducts derivatives. Data points represent the means of triplicate determinations from a representative experiment performed on 3 separate occasions.

Characterization of MPO-dependent formation of isoLG protein adducts in LDL
To further characterize the MPO-mediated mechanism for generating isoLG protein adducts in oxidized LDL, we incubated LDL alone, with the complete MPO-H2O2-NO2- oxidation system, with the complete system plus BHT, or in the presence of the complete system minus each component. All reactions were performed in the presence of the metal chelator DTPA and used Chelex-treated buffer to exclude the possibility of transition metal-catalyzed formation of isoLGs. The oxidation system without MPO or G-GOx failed to produce significant levels of isoLGE2 or iso[4]LGE2 protein adducts, as well as HNE protein adducts (Fig. 4 ). Levels of all three protein adducts were significantly decreased in the absence of NO2-. Addition of BHT to the complete oxidation system inhibited formation of the protein/lipid adducts.



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  		Figure 4. Analysis ofisoLGE2 (A), iso[4]LGE2 (B), and HNE (C) protein adduct formation in LDL exposed to the MPO/H2O2/NO2- system. LDL was incubated in at 37°C for 20 h with isolated human MPO (30 nM), glucose (100 µM), glucose oxidase (20 ng/mL) and NO2- (50 µM) in sodium phosphate buffer (50 mM, pH 7) supplemented with DTPA (200 µM). Additions or deletions to the complete system were as indicated. Butylated hydroxytoluene (BHT, 100 µM) was added to the reaction mixtures as indicated. Data points represent the means ± SD of triplicate determinations from a representative experiment performed on 3 separate occasions.

MPO promotes the formation of isoLG protein adducts in LDL at physiologically relevant levels of NO2-
To investigate the possible role of MPO in formation of isoLG-derived, protein-bound adducts under inflammatory conditions in vivo, we performed LDL oxidation in the physiological range of nitrite concentrations. Levels of NO2- are between 0.5 and 3.6 µM in the plasma of healthy human subjects (48 , 49) , 30–210 µM in saliva, and 0.4–60 µM in gastric juice (50) . At sites of inflammation, nitrite concentration can reach the 50–100 µM range (51 , 52) . Therefore, we exposed LDL to the MPO/G-GOx oxidation system in the presence of 0–100 µM NO2-. MPO-mediated oxidation of LDL generated isoLGE2 and iso[4]LGE2 protein adducts (Fig. 5 A, B), as well as HNE protein adducts (Fig. 5C ), in the presence of pathophysiological plasma levels of NO2-.



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  		Figure 5. NO2- concentration dependence of MPO-dependent A) isoLGE2, B) iso[4]LGE2, and (C) HNE protein adducts and formation. The levels of isoLGs and HNE adducts were determined by ELISA. Data points represent the means ± SD of triplicate determinations from a representative experiment performed on 2 separate occasions.

Isolevuglandin protein adducts are formed in MPO-oxidized, but not in Cu2+-oxidized, LDL in the presence of serum constituents
Recently Dabbagh and Frei (53) showed that serum proteins chelate free transition metal ions and thereby abort lipid peroxidation initiated by free copper ion or free iron-dependent mechanisms. To further explore the potential physiological relevance of MPO as a plausible free radical induction mechanism for generating isoLGs in vivo, we exposed LDL to either the MPO/G-GOx/NO2- or Cu2+ oxidation system in the presence vs. the absence of lipoprotein-deficient serum (LPDS) and examined the reaction mixture for the presence of isoLGE2 protein adducts. Cu2+-catalyzed oxidation of LDL produced protein–lipid adducts alone, but failed to generate significant levels of isoLGE2 protein adducts in the presence of only 10% of LPDS (Fig. 6 ). In marked contrast, MPO-induced formation of isoLGE2 protein adducts was only modestly attenuated by the presence of LPDS (Fig. 6) . Taken together, these results strongly suggest that MPO may serve as a plausible mechanism for the promotion of free radical-induced oxidative generation of isoLG protein adducts in vivo.



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  		Figure 6. LDL oxidation by the MPO/H2O2/NO2- system in the presence of lipoprotein-deficient serum (LPDS, 10%, v/v). The levels of isoLG protein adducts were determined by ELISA. Data points represent the means of duplicate determinations from a representative experiment performed on 3 separate occasions.

Increased levels of isoLG protein adducts, but not F2 isoprostanes, are observed in the Candida sepsis animal model
We next tested the hypothesis that plasma levels of isoLG protein adducts can be used as a biomarker of oxidant stress and inflammation by using an animal model. We chose a Candida sepsis model since we had previously shown that MPO is catalytically active in this model, as monitored by chlorotyrosine formation (39 , 41) , and lipid peroxidation is enhanced within recovered peritoneal lavage fluids after i.p. injection of yeast spore coat (31) . Prior characterization of this model demonstrated comparable peritoneal lavage leukocyte cell number and differential within the first 48 h in wild-type vs. MPO knockout mice after i.p. inoculum and comparable production of superoxide from recovered leukocytes (39 , 41) . Comparable levels of lipoxygenase 12 and cyclooxygenases 1 and 2 in elicited peritoneal lavage cells recovered from wild-type vs. MPO knockout mice have recently been reported (39) , making this model ideal for the proposed studies.

In initial studies, plasma was recovered from wild-type mice at baseline and 12 h after i.p. inoculum of C. albicans. Plasma levels of isoLGE2 protein adducts demonstrated 2.6-fold increases (2.9±0.4 pmol/mg vs. 1.1±0.3 pmol/mg), and plasma levels of iso[4]LGE2 protein adducts demonstrated 3.8-fold (28.4±10.7 pmol/mg vs. 107.1±34.6 pmol/mg) increases, in Candida-infected animals compared with controls (Fig. 7 A, B). Surprisingly, parallel analyses of plasma lipid extracts for total F2 isoprostane content revealed no significant differences in baseline vs. infected animals (Fig. 7C ). Analyses of plasma levels of total F2 isoprostanes at later times post-Candida challenge (24 h, 48 h) demonstrated comparable results, with no significant increases in plasma levels noted (P>0.5 for all comparisons, only data for 48 h post-challenge shown; Fig. 7D ). Thus, Candida sepsis induces enhanced oxidant stress, which was detected by increased systemic levels of isoLG protein adducts, but not F2 isoprostanes. The present studies thus suggest that isoLG protein adducts may serve as useful tools for evaluating oxidant stress at sites of inflammation.



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  		Figure 7. Levels of A) isoLGE2 protein adducts, B) iso[4]LGE2 protein adducts, and C, D) F2 isoprostanes in mouse plasma with and without Candida-induced sepsis. A–C) Plasma was collected 12 h after infection. D) Plasma levels of F2 isoprostanes 48 h after Candida challenge are also shown. Levels of isoLGs were determined by ELISA, and levels of F2 isoprostanes by LC/ESI/MS/MS, as described under Materials and Methods. The statistical significance of the difference between the means of the 2 sets of samples was determined by t test.

MPO participates in the generation of isoLG protein adducts in vivo
To further define the role of MPO in promoting the formation of isoLGs in vivo under inflammatory conditions and further investigate differences between alterations in levels of iso[4]LGE2 protein adducts and F2 isoprostanes during inflammation, additional studies were performed. Levels of iso[4]LGE2 protein adducts and F2 isoprostanes in plasma from wild-type and MPO knockout mice were examined before and after Candida challenge (Fig. 8 ). Iso[4]LGE2 protein adducts were monitored since these species are only produced as a result of free radical mediated oxidation and are not generated by cyclooxygenase, whereas some forms of isoLGE2 protein adducts theoretically are (54) . At baseline, wild-type (n=8) and MPO knockout (n=8) mice were found to possess comparable plasma levels of iso[4]LGE2 protein adducts (Fig. 8A ). Similarly, comparable levels of F2 isoprostanes were noted in plasma recovered from wild-type and MPO knockout mice at baseline (Fig. 8B ). After i.p. injection with C. albicans, plasma levels of iso[4]LGE2 protein adducts were substantially increased in both sets of mice. However, a significant 34% reduction in protein/lipid adduct content was noted in plasma proteins recovered from MPO knockout mice (P=0.003; Fig. 8A ). In contrast, plasma levels of F2 isoprostanes neither increased nor demonstrated differences between wild-type and MPO knockout mice after challenge with C. albicans (Fig. 8B ), despite marked leukocyte recruitment in peritoneal lavage and significant leukocyte elevations within infected mice.



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  		Figure 8. Generation of iso[4]LGE2 protein adducts and F2 isoprostanes in wild-type and MPO knockout mice in the Candida sepsis model. Plasma was collected at baseline and 28 h after infection. The levels of iso[4]LGE2 protein adducts and F2 isoprostanes in plasma were determined by ELISA and stable isotope dilution LC/ESI/MS/MS, respectively. Statistical significance of difference between the means of sets of samples was determined by t test.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present studies provide unambiguous evidence that isoLG protein adducts serve as systemic markers for oxidant stress and are formed in part through MPO-dependent pathways in vivo. IsoLGs are a family of highly reactive lipid peroxidation products produced through the isoprostane pathway in vivo (46) . The term isoLGs refers collectively to all 64 stereo and structural isomers that could be formed by rearrangement of isoprostane endoperoxides. IsoLGs modify (55) and cross-link proteins (56 , 57) , and these modifications have been shown to be associated with chronic inflammation and cardiovascular disease in humans (30) . Whether they participate in the pathogenesis of these disorders is unknown. In contrast to free F2 isoprostanes, which are cleared from the circulation very rapidly (58) , isoLG protein adducts accumulate over the lifetime of the proteins. Since many plasma proteins have half-lives of several weeks and even longer (59) , isoLG protein adducts may provide a cumulative index, like a dosimeter, for oxidative injury. Understanding the physiological pathways involved in formation of isoLG protein adducts is essential for the rational development of antioxidant strategies to prevent their formation. Previous studies demonstrated that MPO-generated reactive nitrogen species could promote lipid peroxidation under physiological conditions (60 , 61) , and studies with MPO knockout mice and chemical peritonitis models demonstrate that this pathway participates in the initiation of lipid peroxidation in vivo (31) . The present studies demonstrate that MPO serves as an enzymatic catalyst for the generation of isoLG protein adducts during Candida sepsis and, vida infra, under inflammatory conditions in vivo.

Prior mass spectrometry studies of formation of isoLGs used free metal ion-induced oxidation of free arachidonic acid (25 , 29) . Since most arachidonic acid in vivo is present in the form of phospholipid esters, we concentrated on the formation of phospholipid-bound isoLGs in lipid vesicles, lipoproteins, and plasma total lipids. Our in vitro and animal model system studies confirm a role for MPO in the formation of isoLGs in vivo. Studies using in vitro models systems confirmed that addition of serum constituents block free metal ion catalyzed isoLG formation in contrast to virtually no effect on the MPO-H2O2-NO2- system. The metal ion independence of the MPO-induced formation of isoLGs is noteworthy in view of the discovery that Cu2+-induced lipid peroxidation is abolished by the presence of plasma proteins (53) . The LC/ESI/MS/MS analyses developed allowed us to directly follow the formation of isoLGs with minimal derivatization procedures. Moreover, they permitted us to distinguish between iso[4]LGE2 and isoLGE2 in the oxidation product mixture using unique transitions characteristic for each structural isomer.

To study the process of isoLG generation in biological systems, we first examined oxidative stress in an animal model where levels of isoLG protein adducts could be detected in plasma and modulated in response to inflammation. We used the Candida sepsis model in mice for several reasons. First, as noted above, we have extensively characterized this model and shown that MPO is catalytically active in this model, as shown by mass spectrometry-based chlorotyrosine determinations (39) . Comparable cell count, differentials, agonist-stimulated superoxide production, and levels of lipid peroxidation catalyzing enzymes (cyclooxygenases 1 and 2, lipoxygenase 12) have been shown using wild-type and MPO knockout mice (31 , 39) , and were again confirmed in control studies (Materials and Methods). Second, as reported earlier, MPO-deficient mice cannot effectively kill Candida infection, so the inflammatory state is more severe (persistent) in such animals (41 , 43) . Therefore, this model provides a situation that is biasing against our hypothesis that we would find fewer isoLG protein adducts in MPO knockout animals. We found that levels of isoLG protein adducts in plasma were increased by at least 2.5-fold in response to Candida-induced inflammation. The present findings thus strongly support the contention that isoLGs are good biomarkers of oxidant stress.

Remarkably, in contrast to isoLG protein adducts, plasma levels of F2 isoprostanes failed to increase substantially in this model in both wild-type and MPO knockout mice. This observation underscores a dichotomy in the behavior of F2 isoprostanes vs. isoLGs in vivo. Although free F2 isoprostanes are chemically stable species that can elicit biological responses by masquerading as prostaglandins, their lifetime in vivo is brief owing to rapid metabolism and excretion. In contrast, LGs and isoLGs are so reactive that they immediately modify proteins. The resulting covalent adducts may accumulate for days and even weeks depending on the lifetime of the target protein, providing a convenient dosimeter of oxidative stress. Although the biological consequences of protein modification by LGs and isoLGs remain largely unexplored, previous in vitro experiments revealed several potentially pathological biological activities. LGs covalently modify LDL, generating a form that undergoes scavenger receptor-mediated recognition and endocytosis by macrophage cells (62) . It is thus conceivable that modification of LDL by isoLGs contributes to foam cell formation and cholesterol accumulation in cells of the artery wall. Protein–protein (57) and DNA–protein cross-links (63) can also be formed after exposure to LGs and isoLGs, processes that may contribute to the cytotoxicity of these species to cultured cells (63 , 64) and to brain tissue in vivo (65) .

Levels of iso[4]LGE2 protein adducts, free radical-induced lipid oxidation products that cannot be formed by cyclooxygenase pathways, were significantly decreased in MPO-deficient animals. These data provide a basis for concluding that MPO is implicated in the formation of isoLG protein adducts in vivo under inflammatory conditions. The reductions noted in MPO knockout mice were only partial, demonstrating that pathways alternative to MPO participate in generation of isoLG protein adducts in vivo. Alternative enzymatic participants in initiation of lipid peroxidation, such as 12 lipoxygenase, COX 1 and COX 2, may contribute to generation of these species. Further, alternative reactive oxidant species capable of initiating lipid peroxidation, such as peroxynitrite formed from superoxide and nitric oxide, likely play a role in formation of the lipid protein adducts.

Inhibition of the multitude of pathways and species capable of promoting oxidative modification of targets at sites of inflammation, each with its own chemical reactivities, represents a formidable challenge. Indeed, it seems highly unlikely that use of a single antioxidant agent will suffice in suppression of the various participants involved. It is therefore of interest that recent studies demonstrate hydroxymethyl glutaryl CoA inhibitors, also known as statins, promote potent systemic antioxidant actions induced via multiple distinct pathways (66 , 67) . The mechanism(s) involved appear not to be due to interception and scavenging of reactive oxidant species like a typical antioxidant, but rather through suppression of generation of free radicals and reactive oxidant species. Statin-induced inhibition in isoprenylation leads both to decreased superoxide formation and enhanced production of nitric oxide (68 69 70) , processes that collectively lead to systemic antioxidant effects (66 , 57) . Interventions aimed at preventing formation of reactive oxidant species through inhibition of their catalytic sources represent a rational strategy for promoting a systemic antioxidant effect. The present studies provide a further rationale for the generation of inhibitors of MPO, agents likely to promote anti-inflammatory and antioxidant actions in vivo.


   ACKNOWLEDGMENTS
 
Supported by National Institutes of Health grants HL70621, HL62526, and HL61878 (S.L.H.) and GM21249 (R.G.S.). Mass spectrometry analyses were performed at the Cleveland Clinic Foundation Mass Spectrometry Core Facility, within the Center for Cardiovascular Diagnostics and Prevention, with the support of GCRC grant # M01 RR018390. M.L.B. is a recipient of a National Institutes of Health NRSA Fellowship. R.Z. is a recipient of an American Heart Association Fellowship.


   FOOTNOTES
 
doi: 10.1096/fj.03-0086com

Received for publication February 18, 2003. Accepted for publication August 5, 2003.


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M. Navab, G. M. Ananthramaiah, S. T. Reddy, B. J. Van Lenten, B. J. Ansell, G. C. Fonarow, K. Vahabzadeh, S. Hama, G. Hough, N. Kamranpour, et al.
Thematic review series: The Pathogenesis of Atherosclerosis The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL
J. Lipid Res., June 1, 2004; 45(6): 993 - 1007.
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