Human phagocytes employ the myeloperoxidase-hydrogen peroxide system to synthesize dityrosine, trityrosine, pulcherosine, and isodityrosine by a tyrosyl radical-dependent pathway.

Myeloperoxidase, a heme protein secreted by activated phagocytes, may be a catalyst for lipoprotein oxidation in vivo. Active myeloperoxidase is a component of human atherosclerotic lesions, and atherosclerotic tissue exhibits selective enrichment of protein dityrosine cross-links, a well characterized product of myeloperoxidase. Tyrosylation of lipoproteins with peroxidase-generated tyrosyl radical generates multiple protein-bound tyrosine oxidation products in addition to dityrosine. The structural characterization of these products would thus serve as an important step in determining the role of myeloperoxidase in lipoprotein oxidation in the artery wall. We now report the identification and characterization of four distinct tyrosyl radical addition products generated by human phagocytes. Activated neutrophils synthesized three major fluorescent products from L-tyrosine; on reverse phase HPLC, each compound coeluted with fluorescent oxidation products formed by myeloperoxidase. We purified the oxidation products to apparent homogeneity by cation and anion exchange chromatographies and identified the compounds as dityrosine (3,3′-dityrosine), trityrosine (3,3′,5′,3″-trityrosine) and pulcherosine (5-[4″-(2-carboxy-2-aminoethyl)phenoxy]3,3′-dityrosine) by high resolution NMR spectroscopy and mass spectrometry. Additionally, we have found that dityrosine is a precursor to trityrosine, but not pulcherosine. In a search for a precursor to pulcherosine, we identified isodityrosine (3-[4′-(2-carboxy-2-aminoethyl)phenoxy]tyrosine), a non-fluorescent product of L-tyrosine oxidation by human phagocytes. Our results represent the first identification of this family of tyrosyl radical addition products in a mammalian system. Moreover, these compounds may serve as markers specific for tyrosyl radical-mediated oxidative damage in atherosclerosis and other inflammatory conditions.

Myeloperoxidase, a heme protein secreted by activated phagocytes, may be a catalyst for lipoprotein oxidation in vivo. Active myeloperoxidase is a component of human atherosclerotic lesions, and atherosclerotic tissue exhibits selective enrichment of protein dityrosine cross-links, a well characterized product of myeloperoxidase. Tyrosylation of lipoproteins with peroxidasegenerated tyrosyl radical generates multiple proteinbound tyrosine oxidation products in addition to dityrosine. The structural characterization of these products would thus serve as an important step in determining the role of myeloperoxidase in lipoprotein oxidation in the artery wall. We now report the identification and characterization of four distinct tyrosyl radical addition products generated by human phagocytes. Activated neutrophils synthesized three major fluorescent products from L-tyrosine; on reverse phase HPLC, each compound coeluted with fluorescent oxidation products formed by myeloperoxidase. We purified the oxidation products to apparent homogeneity by cation and anion exchange chromatographies and identified the compounds as dityrosine (3,3-dityrosine), trityrosine (3,3,5,3؆-trityrosine) and pulcherosine (5-[4؆-(2carboxy-2-aminoethyl)phenoxy]3,3-dityrosine) by high resolution NMR spectroscopy and mass spectrometry. Additionally, we have found that dityrosine is a precursor to trityrosine, but not pulcherosine. In a search for a precursor to pulcherosine, we identified isodityrosine (3-[4-(2-carboxy-2-aminoethyl)phenoxy]tyrosine), a nonfluorescent product of L-tyrosine oxidation by human phagocytes. Our results represent the first identification of this family of tyrosyl radical addition products in a mammalian system. Moreover, these compounds may serve as markers specific for tyrosyl radical-mediated oxidative damage in atherosclerosis and other inflammatory conditions.
Oxidized low density lipoprotein (LDL) 1 may play a pivotal role in the pathogenesis of atherosclerosis (1)(2)(3)(4), but the mechanisms that damage LDL in vivo have yet to be identified. A potential pathway involves myeloperoxidase, a heme protein secreted by neutrophils and monocytes (4). Phagocytes employ the myeloperoxidase-H 2 O 2 system to generate potent cytotoxins that kill invading pathogens and tumor cells (5)(6)(7)(8); under pathological conditions, normal tissue may also be a target for damage. Active myeloperoxidase is a component of human atherosclerotic lesions, where it co-localizes with monocytes and macrophages (9). The pattern of immunostaining for the enzyme in human atherosclerotic lesions (9) is remarkably similar to that for protein-bound lipid oxidation products in rabbit lesions (10), implicating myeloperoxidase as a potential pathway for LDL oxidation in vivo.
Hypochlorous acid is the best characterized product of myeloperoxidase (11), which is the only human enzyme known to produce this oxidant under physiological conditions (12). Hypochlorous acid is a potent cytotoxin that oxidatively bleaches heme proteins (13) and chlorinates proteins (14,15) and unsaturated lipids (16,17). LDL modified with reagent hypochlorous acid undergoes aggregation and causes macrophages to accumulate cholesteryl ester (18); lipoproteins with similar properties have been isolated from atherosclerotic lesions (19 -21). A monoclonal antibody specific for hypochlorous acid-modified proteins recognizes epitopes in human atherosclerotic lesions, as well as LDL-like particles isolated from aortic tissue, strongly suggesting that myeloperoxidase is promoting oxidation in the artery wall (22).
Tyrosine oxidation products, including dityrosine (30,31), pulcherosine (32), isodityrosine (33), and trityrosine (30,32), are a common post-translational modification found in bacteria (34), yeast (35,36), plants (33), and metazoans (30 -33, 37). In * This work was supported in part by a grant-in-aid from the American Heart Association and by National Institutes of Health Grants RO1 HD29920 and RO1 AG12293. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  all cases where the biochemical pathway is known, the mechanism involves direct oxidation of protein tyrosyl residues by a heme protein (30 -33, 35, 36). In contrast, myeloperoxidasecatalyzed formation of protein dityrosine cross-links requires free L-tyrosine (28,29), implying that tyrosyl radical generated by the enzyme is acting as a diffusible radical catalyst. High density lipoprotein exposed to peroxidase-generated tyrosyl radical undergoes tyrosylation and cross-linking of its apolipoproteins by a tyrosine-dependent reaction (27). Examination of acid hydrolysates of high density lipoprotein exposed to L-[ 14 C]tyrosine, H 2 O 2 , and peroxidase revealed that dityrosine accounted for only 30% of radiolabel incorporated into the protein. Moreover, multiple L-[ 14 C]tyrosine-labeled peaks were present on cellulose phosphate chromatography, suggesting that other products might be formed by tyrosyl radical (27).
We have proposed that dityrosine may serve as a marker for proteins oxidatively damaged by the myeloperoxidase-H 2 O 2 system of activated phagocytes (23,(27)(28)(29). Recent studies indicate that levels of protein-bound dityrosine are elevated in human atherosclerotic lesions, raising the possibility that tyrosyl radical is a catalyst for LDL oxidation in vivo. 2 To search for compounds that might serve as additional markers of phagocyte-mediated protein damage, we examined the ability of myeloperoxidase to catalyze the one electron oxidation of Ltyrosine. We show that human neutrophils employ the myeloperoxidase-H 2 O 2 system to generate a family of tyrosyl radical addition products that are acid-stable and strongly fluorescent, making them potentially useful markers for phagocyte-mediated damage in vivo.

Materials
Glucose oxidase and crystalline catalase (from beef liver; thymolfree) were purchased from Boehringer Mannheim. L-[ 13 C 6 ]Tyrosine (Ͼ99% pure) was from Isotec. All other reagents were from either Sigma or Fisher Scientific unless otherwise indicated.

Methods
Isolation of Myeloperoxidase-Nonidet P-40-treated leukocytes obtained by leukopheresis from a patient with chronic myelogenous leukemia (39) were extracted with cetyltrimethylammonium bromide (40). Myeloperoxidase was then isolated by sequential lectin-affinity chromatography, ammonium sulfate precipitation, and gel filtration chromatography as described previously (23,40). Myeloperoxidase concentration was determined spectrophotometrically (⑀ 430 ϭ 170 mM Ϫ1 cm Ϫ1 ; Ref. 41) and the isolated enzyme had an A 430 /A 280 ratio of 0.71. L-Tyrosine Oxidation by Myeloperoxidase-Reactions were carried out in buffer A (50 mM phosphate, 100 M DTPA, pH 7.4), which had been passed over a Chelex-100 column (Bio-Rad) to remove metal ions. The reaction mixture contained a final concentration of 2 mM L-tyrosine, 4 nM myeloperoxidase, 1 mg/ml glucose, and 50 ng/ml glucose oxidase. After a 2-h incubatation at 37°C, the reaction was terminated by addition of catalase (400 nM) and the reaction mixture was stored at Ϫ20°C until analysis.
Isolation of L-Tyrosine Oxidation Products by Ion Exchange Chromatography-Reaction mixture (1 liter) was acidified to pH 3.0 with HCl and subjected to strong cation exchange chromatography (AG-50WX2; Bio-Rad) as a desalting step. The column was eluted with 2 M NH 4 OH. The resulting amino acid solution was lyophilized, dissolved in buffer B (10 mM acetic acid, pH 3.0), and then subjected to fast protein liquid chromatography (FPLC) on a Pharmacia Mono S HR 5/5 column equilibrated with buffer B. Amino acids were eluted from the cation exchange column with a 60-ml linear gradient of 0 -0.25 M NaCl in buffer B at a flow rate of 1 ml/min. Collected peaks were desalted as above, dissolved in buffer C (10 mM Tris base, pH 9.0), subjected to FPLC on a Pharmacia Mono Q HR 5/5 anion exchange column equilibrated with buffer C, and eluted with a 60-ml linear gradient of 0 -0.25 M NaCl in buffer C at 1 ml/min. Each amino acid was then desalted and again subjected to cation exchange chromatography as described above.
L-Tyrosine Oxidation by Activated Human Neutrophils-Neutrophils were prepared from human blood anticoagulated with EDTA (5.4 mM) using Polymorph-Prep (Nycomed, Sunnyvale, CA) as described previously (23). Freshly harvested cells were resuspended in medium A (Ca 2ϩ -, Mg 2ϩ -, and phenol red-free Hank's balanced salt solution, pH 7.4; Life Technologies, Inc.) and used immediately for experiments. Reactions were carried out at 37°C in medium A supplemented with 1 mM L-tyrosine, 0.1 mM DTPA, and 1 mg/ml D-glucose. Neutrophils (5 ϫ 10 5 cells/ml) were activated by the addition of of 200 nM phorbol 12myristate 13-acetate (PMA; dissolved in dimethyl sulfoxide) and were maintained in suspension by periodic inversion. Reactions were stopped by the addition of 400 nM catalase, the cells were pelleted by centrifugation, and the supernatant was stored at Ϫ20°C until analysis. Superoxide (O 2 . ) production by neutrophils was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome c (23).

Reverse Phase HPLC Analysis of Fluorescent Neutrophil and Myeloperoxidase Reaction
Products-Oxidation reactions were filtered (Acrodisc LC PVDF, 0.2 m; Gelman Sciences) and analyzed on a Beckman Ultrasphere ODS column (250 ϫ 4.6 mm, 5-m diameter particles). Products were eluted isocratically at 2 ml/min with a mobile phase of 0.1 M Na 2 HPO 4 , 0.1 mM EDTA adjusted to pH 5.9 with acetic acid. L-tyrosine was detected by absorbance (280 nm) using a Beckman model 163 variable wavelength detector. Fluorescent compounds were detected with a Waters model 470 scanning fluorescence detector ( ex 296 nm, em 414 nm).
NMR Spectroscopy-One-and two-dimensional NMR spectra were acquired at 25°C using a Varian Unity 500 spectrometer (499.843 MHz for 1 H) interfaced to a Sun SPARC-2 workstation and equipped with a Nalorac triple-resonance indirect-detection probe. Prior to analysis, FPLC-purified products were dissolved in D 2 O (Cambridge Isotope Laboratories). Proton and carbon-13 chemical shifts were referenced to external sodium 3-(trimethylsilyl)propionate-2,2,3,3-d 4 in D 2 O. For two-dimensional experiments, data were acquired in the hypercomplex mode (42).
Gas Chromatography-Mass Spectrometry (GC-MS)-FPLC-purified and desalted oxidation products were evaporated under N 2 and esterified by incubation with 200 l of HBr (3.5 M) in n-propanol (Cambridge Isotope Laboratories) at 100°C for 30 min. Pentafluoropropyl derivatives were then prepared by incubation of dried propylated amino acids with 50 l of ethyl acetate/pentafluoropropionic acid anhydride (4:1, v/v) at 60°C for 30 min. Derivatized amino acids were stored in reagent solution until analysis.
GC and direct probe MS analyses were obtained with a HP-5988A mass spectrometer equipped with a Technivent Vector 1 Data System. Capillary GC was performed using a DB-1 column (8 m, 0.33 mm inner diameter, 1-m film thickness; J & W Scientific) in the splitless injection mode. The initial column temperature of 70°C was increased to 150°C at 60°C/min, and then to 250°C at 10°C/min. The injector, transfer line, and ion source temperature were set at 220°C, 250°C, and 150°C, respectively. Mass spectra were obtained in the negativeion chemical ionization mode using methane as the reagent gas.

Myeloperoxidase Generates a Family of Tyrosyl Radical
Addition Products-Three major fluorescent peaks, termed compounds I, II, and III, were identified by Mono S chromatography when L-tyrosine was oxidized by the myeloperoxidase-H 2 O 2 system (Fig. 1). Production of all three oxidation products required active myeloperoxidase and the H 2 O 2 generating system and was inhibited by catalase (a scavenger of H 2 O 2 ), azide (a heme poison), and ascorbate (which reacts with both H 2 O 2 and tyrosyl radical). Compounds I, II, and III eluted from a Mono S cation exchange column at 60 mM, 115 mM, and 105 mM NaCl, respectively; compound I coeluted with authentic dityrosine. Each of the products exhibited dityrosine-like fluores-cence ( ex 315 nm and em 410 nm, respectively, pH 9.0) and tyrosine-like absorbance ( max , ϳ280 nm, pH 3.0). Each fluorescent peak was collected, desalted and subjected to chromatography on a Mono Q column as described under "Methods." Compounds I, II, and III eluted from the anion exchange column at 135, 145, and 180 mM NaCl, respectively. The purified oxidation products employed for the structural studies described below were apparently homogeneous as assessed by repeat Mono S chromatography as well as by reverse phase HPLC; each compound eluted as a single, symmetrical peak as monitored by both fluorescence and absorbance.
Structural Characterization of the Fluorescent L-Tyrosine Oxidation Products Generated by Myeloperoxidase-1 H NMR resonances from the aromatic region (6.8 -7.4 ppm) of compounds I, II and III and their structural assignments are shown in Fig.  2. This region of compound I ( Fig. 2A) was very similar to that previously reported for 3,3Ј-dityrosine isolated from yeast spore coats (32)  To confirm the assignments of the protons in compound II, we used total correlation spectroscopy to examine the connectivities of the protons (Fig. 3). Proton-proton correlations between the single proton peaks at 6.96 (H a ) and 7.23 ppm (H b ), and the two proton peaks at 7.02 (H f ) and 7.28 ppm (H g ), indicate that the protons are adjacent to one another on the aromatic ring. Correlation between peaks at 7.01 (H e ) and 7.12 ppm (H d ) and the peaks at 7.23 (H b ) and 7.32 ppm (H c ) are indicative of four bond, long-range interactions. These data are consistent with the structure of pulcherosine.
Mass spectrometry was used to confirm the proposed structures of compounds I, II and IlI. The n-propyl ester, pentafluoropropyl derivative of each compound was prepared, and the dityrosine and pulcherosine derivatives subjected to GC-MS in the negative-ion chemical ionization mode. Because it was rel-atively non-volatile, derivatized trityrosine was analyzed using direct probe MS. Dityrosine exhibited a low abundance ion peak at mass-to-charge (m/z) 1028, the calculated molecular in buffer A (1 liter) was incubated with myeloperoxidase (4 nM) and an H 2 O 2 generating system (100 ng/ml glucose oxidase and 0.1 mg/ml D-glucose). After a 2-h incubation at 37°C, the reaction was terminated by the addition of catalase (400 nM). The reaction mixture was desalted and subjected to FPLC on a Mono S HR 5/5 column as described under "Methods." The peaks of material labeled I, II, III, and IV were purified to apparent homogeneity by sequential chromatography on anion exchange (Mono Q HR5/5) and cation exchange (Mono S HR5/5) columns, and subsequently identified as dityrosine, pulcherosine, trityrosine, and isodityrosine, respectively, in the structural studies described under "Results." 1541(M Ϫ ), as well as major ions expected from loss of HF and CF 3 CF 2 CHO at m/z 1521 and 1393, respectively. The mass spectra of compounds I, II, and III are thus in excellent agreement with the structures determined using multinuclear NMR spectroscopy.

Ultraviolet Absorption and Fluorescence Spectra with Molar Extinction Coefficient Determinations of the Major Tyrosyl Radical Addition
Products-Radiolabeled dityrosine, pulcherosine, and trityrosine were isolated by FPLC from an L-[ 14 C]tyrosine solution of known specific activity oxidized with myeloperoxidase, and their concentrations determined by scintillation spectrometry. The extinction coefficients, ultraviolet absorption spectra, and fluorescence excitation/emission spectra of the compounds were then determined at acid and alkaline pH. These data, which are in good agreement with previously reported results for tyrosine oxidation products isolated from sea urchins, insects, and yeast (24,25,32,36), are summarized in Table I.
Isolation and Identification of Isodityrosine, a Non-fluorescent L-Tyrosine Radical Addition Product Generated by Myeloperoxidase-Initial fractionation of the myeloperoxidase-Ltyrosine-H 2 O 2 reaction mixture revealed a small shoulder of material, which eluted just after dityrosine on cation exchange chromatography ( Fig. 1, peak IV). This material exhibited tyrosine-like absorbance but not dityrosine-like fluorescence. To investigate the structure of this compound, L-[ 13 C 6 ]tyrosine in buffer A was exposed to the myeloperoxidase-H 2 O 2 system, and the product was isolated by sequential cation, anion, and cation exchange FPLC as described above for compounds I-III. The isolated compound, which eluted as a single, symmetrical peak on reverse phase HPLC analysis, was subjected to heteronuclear single quantum coherence NMR spectroscopy (Fig. 4). Isodityrosine might be an intermediate in the synthesis of pulcherosine by myeloperoxidase. To test this hypothesis, we supplemented the complete myeloperoxidase-H 2 O 2 system with 1 mM L-tyrosine and 0.02 mM isodityrosine, and then monitored the formation of pulcherosine. The yield of pulcherosine increased 2-fold in the presence of isodityrosine whereas the formation of trityrosine was unaffected. In contrast, the presence of dityrosine (0.02 mM) caused a 3-fold increase in the yield of trityrosine, but did not affect the yield of pulcherosine. These results strongly suggest that isodityrosine and dityrosine are intermediates in the formation of pulcherosine and trityrosine, respectively.

Neutrophils Employ the Myeloperoxidase-H 2 O 2 System to Convert L-Tyrosine to Dityrosine, Trityrosine, Pulcherosine, and
Isodityrosine-To explore the possibility that the family of tyrosyl radical addition products generated by myeloperoxidase are indices of phagocyte activation, we used reverse phase HPLC with fluorescence monitoring to quantify the synthesis of each compound by phorbol ester-stimulated human neutrophils. Cells were incubated at 37°C in a physiological salt solution supplemented with 1 mM L-tyrosine and 0.1 mM DTPA. Three peaks with dityrosine-like fluorescence were observed that co-migrated with authentic dityrosine, trityrosine, and pulcherosine generated by the myeloperoxidase-H 2 O 2 system (Fig. 5). The identity of the neutrophil-generated fluorescent compounds was confirmed by demonstrating co-migration with each myeloperoxidase-generated oxidation product on two independent reverse phase HPLC systems (43) and on anion and cation exchange FPLC. The non-fluorescent product, isodityrosine, was readily identified by GC-MS analysis, but was below the UV detection limit on HPLC.
Production of dityrosine, trityrosine, and pulcherosine required phagocytes, L-tyrosine, and activation of the cells by phorbol ester (Table II). L-Tyrosine oxidation by the cells was independent of iron or copper because the buffer contained DTPA, a potent inhibitor of metal-catalyzed reactions (44). As with the reaction catalyzed by purified myeloperoxidase, the neutrophil reaction was inhibited by catalase, by the heme poisons azide and cyanide, and by ascorbate (Table II). Collectively, these results suggest that the family of tyrosyl radical addition products generated by myeloperoxidase constitutes molecular markers of phagocyte activation.
Dityrosine Promotes the Synthesis of Trityrosine by Neutrophils-Studies with isolated myeloperoxidase suggested that isodityrosine and dityrosine were precursors for pulcherosine and trityrosine, respectively. To test the hypothesis that dityrosine was similarly employed by the myeloperoxidase system of activated human neutrophils to generate trityrosine, we supplemented the medium of neutrophils with dityrosine (0.01 mM) and L-tyrosine (1 mM) and then monitored the kinetics of trityrosine and pulcherosine synthesis. The presence of dity-  rosine led to a decrease in the lag phase for trityrosine synthesis (Fig. 6, upper panel). Moreover, there was a dramatic 8-fold increase in the yield of trityrosine. These results strongly suggest that dityrosine is a precursor in the generation of trityrosine. In contrast to trityrosine, the kinetics and yield of pulcherosine (Fig. 6, lower panel) were unaffected by the presence of dityrosine, suggesting that pulcherosine generation involves another intermediate. These results suggest that ty-rosyl radical reacts by two different pathways to generate trityrosine and pulcherosine.
Superoxide Dismutase Stimulates L-Tyrosine Oxidation by Activated Neutrophils-The initial product of the neutrophil oxidase is O 2 . , the one-electron reduced form of molecular oxygen (45). O 2 . scavenges tyrosyl radical by acting as an electron donor (46,47). This reaction is blocked by superoxide dismutase, which catalyzes the dismutation of O 2 . to H 2 O 2 (46,47).
To explore the role of O 2 . in L-tyrosine oxidation, the kinetics of dityrosine, trityrosine, and pulcherosine generation by activated neutrophils in the presence and absence of superoxide dismutase were monitored. Neutrophil activation resulted in O 2 . production after a lag phase of ϳ2 min (data not shown).
The progress curve of dityrosine production was similarly nonlinear but with a longer lag phase of ϳ6 min (Fig. 7, top panel).
In contrast, the lag phases for O 2 . generation and dityrosine synthesis were virtually identical when superoxide dismutase was included. Catalase inhibited the production of dityrosine, trityrosine, and pulchosine by neutrophils (Fig. 7), confirming that H 2 O 2 is necessary for their synthesis. The progress curves for both pulcherosine and trityrosine production by activated neutrophils exhibited lag phases that were greater than that observed for dityrosine (Fig. 7). As with dityrosine, superoxide dismutase significantly shortened the lag phases for trityrosine and pulcherosine production (Fig. 7,  middle and bottom panels). Superoxide dismutase led to a 1.5-fold increase in dityrosine and 8-and 4-fold increases in trityrosine and pulcherosine, respectively. Superoxide dismutase may have stimulated the yield of L-tyrosine oxidation products by blocking the scavenging of tyrosyl radical by O 2 .  Fig. 1 or with activated human neutrophils as described in the legend to Fig. 6. After a 2-h incubation at 37°C, the reactions were terminated by addition of catalase (400 nM) and subjected to analysis by reverse phase HPLC as described under "Methods." Tyrosine oxidation products were detected by monitoring fluorescence ( ex 296 nm, em 414 nm).

TABLE II
Requirements for dityrosine, trityrosine, and pulcherosine synthesis by neutrophils Oxidation of L-tyrosine by neutrophils (5 ϫ 10 5 cells/ml) was carried out in medium A supplemented with 0.1 mM DTPA, 1 mg/ml D-glucose, and 1 mM L-tyrosine. Neutrophils were activated by the addition of 200 nM PMA. After a 2-h incubation at 37°C, the reaction was stopped by the addition of 400 nM catalase. Samples were analyzed by reverse phase HPLC as described under "Methods." The limit of detection for each compound was less than 1 pmol. Values are expressed as picomoles per 5 ϫ 10 5 cells and are the mean of duplicate determinations. Similar results were found in two independent experiments.

DISCUSSION
Several lines of evidence indicate that human neutrophils employ the myeloperoxidase-H 2 O 2 system to convert L-tyrosine to tyrosyl radical, which then undergoes radical addition reactions to form dityrosine, isodityrosine, trityrosine, and pulcherosine. First, purified myeloperoxidase converted L-tyrosine to three fluorescent products and one non-fluorescent product by a reaction that required H 2 O 2 and was sensitive to inhibition by heme poisons. The products of this reaction were identified as dityrosine, isodityrosine, trityrosine, and pulcherosine by NMR and mass spectroscopy. Second, activation of human neutrophils with phorbol ester led to the formation of the identical family of tyrosyl radical addition products. As with myeloperoxidase, synthesis of the compounds required H 2 O 2 and was inhibited by heme poisons. Finally, the formation of dityrosine, trityrosine and pulcherosine by activated neutrophils was inhibited by ascorbic acid and stimulated by superoxide dismutase. Both ascorbate and O 2 . , the initial product of the neutrophil oxidase (45), are known to repair tyrosyl radical, and the reaction of O 2 . with tyrosyl radical is blocked by superoxide dismutase (46,47). These results represent the first identification of this family of tyrosyl radical addition products in a mammalian system and suggest their potential as specific markers for phagocyte-mediated oxidative damage. The one-electron oxidation of L-tyrosine by myeloperoxidase bears striking parallels with the chemistry of phenoxyl radical (49,50). In both systems, the spectrum of products is readily explained by radical-radical coupling at the predominant sites of electron density on the oxygen and aromatic carbons (Scheme I). Substituents in the para position of phenoxyl radical block both o,pЈ and p,pЈ dimerization (49,50). L-Tyrosine oxidation by myeloperoxidase generates the products expected from para-substituted phenoxyl radical, including compounds with carbon-carbon cross-links (dityrosine, trityrosine, pulcherosine) and carbon-oxygen cross-links (pulcherosine and isodityrosine), strongly implicating tyrosyl radical in the reaction pathway.
Three observations suggest that dityrosine itself might be oxidized by activated neutrophils to dityrosyl radical, which then reacts with tyrosyl radical to form trityrosine. First, the lag phase for the conversion of L-tyrosine to trityrosine by the cells was significantly greater than that observed for dityrosine, suggesting that trityrosine production was dependent upon dityrosine formation. Second, the lag phases for the synthesis of both dityrosine and trityrosine were shortened by superoxide dismutase; this enzyme scavenges O 2 . , which is known to repair tyrosyl radical (46,47). Third, when a low concentration of dityrosine was included together with L-tyrosine in the medium of the cells, the lag phase for trityrosine synthesis was shortened and the yield of the compound was increased 8-fold. In contrast, dityrosine had little effect on either the progress curve or the yield of pulcherosine. Scheme II is consistent with these results and the chemistry of phenoxyl radical (49,50). Oxidation of LDL may represent an important step in conferring atherogenic potential to the modified lipoprotein. However, the biochemical pathways that mediate LDL oxidation in vivo have yet to be identified (1)(2)(3)(4). Previously, we have shown that active myeloperoxidase is expressed in human atherosclerotic lesions (9) and that myeloperoxidase-derived tyrosyl radical initiates LDL lipid peroxidation (29). LDL isolated from vascular tissue exhibits evidence of lipid peroxidation (19 -21) and is aggregated (21), suggesting that tyrosyl radical may account in part for intermolecular cross-linking of artery wall FIG. 7. Progress curves for the synthesis of dityrosine (A), trityrosine (B), and pulcherosine (C) by activated human neutrophils. Neutrophils (5 ϫ 10 5 cells/ml) were incubated at 37°C in medium A supplemented with 1 mM L-tyrosine, 0.1 mM DTPA, and 1 mg/ml D-glucose. Where indicated, superoxide dismutase (sod; 150 nM) or catalase (400 nM) were included in the medium. After stimulation of the cells with 200 nM PMA, aliquots of the medium were removed at the indicated times and subjected to analysis by reverse phase HPLC as described under "Methods." Values are expressed as picomoles per 5 ϫ 10 5 cells and represent the mean of duplicate determinations. SCHEME II. Proposed reaction pathways for the generation of tyrosyl radical addition products by myeloperoxidase. The proposed pathway for trityrosine production involves conversion of dityrosine to dityrosyl radical, which cross-links with a free tyrosyl radical to form trityrosine. Dityrosine might be oxidized directly by myeloperoxidase or indirectly by tyrosyl radical. Indeed, recent kinetic studies demonstrate that dityrosine is an excellent substrate for oxidation by myeloperoxidase (51). In light of the evidence that isodityrosine increases the formation of pulcherosine by myeloperoxidase, it is likely that this compound is similarly an intermediate in pulcherosine formation. Isodityrosine would then be oxidized to a radical which cross-links with tyrosyl radical to generate pulcherosine. lipoproteins. L-Tyrosine oxidation may also play a role in stimulating the cellular uptake of lipoproteins because cross-linking causes LDL to aggregate and stimulates its uptake by macrophages (18,21,22,52).
Tyrosyl radical generated by activated phagocytes causes protein tyrosylation (28), and human atherosclerotic lesions contain elevated levels of protein-bound dityrosine, 2 implicating peroxidative pathways as one mechanism for oxidative damage of the vascular wall. The L-tyrosine oxidation products we have described are stable to acid hydrolysis, intensely fluorescent, and readily detected by mass spectrometry, making them attractive candidates for monitoring protein oxidative damage. Moreover, dityrosine accounts for only a third of the L-tyrosine covalently incorporated into the apolipoproteins of high density lipoprotein exposed to tyrosyl radical (27), supporting our hypothesis that myeloperoxidase-damaged proteins may contain multiple significant L-tyrosine oxidation products. The detection of trityrosine, isodityrosine, and pulcherosine in proteins tyrosylated by myeloperoxidase in vitro, and the demonstration that a similar pattern of protein-bound oxidation products exists in human vascular lesions, would strongly support our hypothesis that tyrosyl radical generated by myeloperoxidase plays a critical role in oxidative damage during atherosclerosis and inflammation.