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J Biol Chem, Vol. 274, Issue 47, 33440-33448, November 19, 1999

Molecular Chlorine Generated by the Myeloperoxidase-Hydrogen Peroxide-Chloride System of Phagocytes Produces 5-Chlorocytosine in Bacterial RNA^*

Jeffrey P. Henderson^§, Jaeman Byun, and Jay W. Heinecke^¶

From the Departments of  Medicine and ^¶ Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Myeloperoxidase, a heme enzyme secreted by activated phagocytes, uses H₂O₂ and Cl to generate the chlorinating intermediate hypochlorous acid (HOCl). This potent cytotoxic oxidant plays a critical role in host defenses against invading pathogens. In this study, we explore the possibility that myeloperoxidase-derived HOCl might oxidize nucleic acids. When we exposed 2'-deoxycytidine to the myeloperoxidase-H₂O₂-Cl system, we obtained a single major product that was identified as 5-chloro-2'-deoxycytidine using mass spectrometry, high performance liquid chromatography, UV-visible spectroscopy, and NMR spectroscopy. 5-Chloro-2'-deoxycytidine production by myeloperoxidase required H₂O₂ and Cl, suggesting that HOCl is an intermediate in the reaction. However, reagent HOCl failed to generate 5-chloro-2'-deoxycytidine in the absence of Cl. Moreover, chlorination of 2'-deoxycytidine was optimal under acidic conditions in the presence of Cl. These results implicate molecular chlorine (Cl₂), which is in equilibrium with HOCl through a reaction requiring Cl and H⁺, in the generation of 5-chloro-2'-deoxycytidine. Activated human neutrophils were able to generate 5-chloro-2'-deoxycytidine. Cellular chlorination was blocked by catalase and heme poisons, consistent with a myeloperoxidase-catalyzed reaction. The myeloperoxidase-H₂O₂-Cl system generated similar levels of 5-chlorocytosine in RNA and DNA in vitro. In striking contrast, only cell-associated RNA acquired detectable levels of 5-chlorocytosine when intact Escherichia coli was exposed to the myeloperoxidase system. This observation suggests that oxidizing intermediates generated by myeloperoxidase selectively target intracellular RNA for chlorination. Collectively, these results indicate that Cl₂ derived from HOCl generates 5-chloro-2'-deoxycytidine during the myeloperoxidase-catalyzed oxidation of 2'-deoxycytidine. Phagocytic generation of Cl₂ therefore may constitute one mechanism for oxidizing nucleic acids at sites of inflammation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Oxidants generated by activated white blood cells play a central role in host antimicrobial defenses but may also damage host tissues (1-5). Oxidant production begins with a membrane-associated NADPH oxidase, which reduces molecular oxygen to superoxide. Dismutation of superoxide then yields H₂O₂. Phagocytes can use the oxidizing capacity of this H₂O₂ to generate potent cytotoxic oxidants because they also secrete the heme enzyme myeloperoxidase. At plasma concentrations of chloride (Cl), the major action of the enzyme is to convert Cl to hypochlorous acid (HOCl) (6, 7).

HOCl undergoes numerous reactions with biomolecules, including aromatic chlorination (8, 9), double bond addition (10, 11), chloramine formation (12-16), aldehyde generation (12, 17, 18), and oxidation of thiols (19). Myeloperoxidase also converts free tyrosine to tyrosyl radical, a reactive intermediate that cross-links proteins and initiates lipid peroxidation (20-23). Recent studies indicate that myeloperoxidase catalyzes the nitrite-dependent nitration of tyrosine (24-26). Mass spectrometric analyses of tissue proteins have detected elevated levels of oxidized amino acids characteristic of these reactions at sites of inflammation, implicating myeloperoxidase as one pathway for oxidative damage in vivo (8, 27-29).

We have previously demonstrated that HOCl generated by myeloperoxidase is in equilibrium with molecular chlorine (Cl₂) through a reaction that requires Cl and H⁺ (30).

In vitro studies suggested that the production of 3-chlorotyrosine by reagent HOCl involves molecular chlorine rather than HOCl itself. Moreover, Cl₂ derived from HOCl converted cholesterol into a battery of chlorinated and oxygenated sterols (9). We recently demonstrated that 3-chlorotyrosine levels are selectively elevated in human atherosclerotic tissue, strongly suggesting that oxidative reactions involving HOCl or its derivatives are physiologically relevant to tissue damage at sites of inflammation (8).

Chronic inflammation is associated with an increased risk of cancer, raising the possibility that reactive intermediates generated by phagocytes might damage nucleic acids in living cells, compromising the integrity of the genome or altering cellular functions (31). Moreover, in vitro studies have identified numerous modified nucleic acids as products of oxidative reactions. Bases in DNA, for example, are hydroxylated by hydroxyl radical and singlet oxygen (32-34). Reactive nitrogen species generate nitrated nucleobases, such as 8-nitroguanine, and deaminated nucleobases, such as xanthine (35-39). Single-strand breaks in DNA have been proposed as a major effect of superoxide damage (40). Halogenated and oxygenated nucleobases are synthesized when high concentrations of hypohalous acid are reacted with free pyrimidines and purines in dilute acid (14-16, 41). 5-Chlorouracil has been detected in acid-hydrolyzed DNA exposed to reagent HOCl at neutral pH (42), although DNA does not contain uracil. Nucleobases also can be modified by aldehydes, which result from lipid peroxidation (43). Oxidized nucleic acids have been detected in DNA extracted from cells exposed to oxidizing conditions and in human urine (44-46). However, the pathways that promote the production of oxidized nucleobases in vivo and their relevance to human pathology are incompletely understood.

We examined the ability of the myeloperoxidase-H₂O₂-Cl system to oxidize nucleic acids by using 2'-deoxycytidine as a target. Using mass spectrometry, chromatography, UV-visible spectroscopy, and NMR spectroscopy, we identified 5-chloro-2'-deoxycytidine as the major stable product. Activated neutrophils were also observed to generate 5-chloro-2'-deoxycytidine in a reaction that was inhibited by heme poisons and catalase. Reactions with reagent HOCl implicated molecular chlorine as the oxidizing intermediate in the reaction pathway. Unexpectedly, 5-chlorocytosine was generated in the RNA but not the DNA of Escherichia coli exposed to the myeloperoxidase-H₂O₂-Cl system. These observations raise the possibility that chlorinating intermediates generated by myeloperoxidase modify nucleic acids of pathogens that are attacked by phagocytes. Such intermediates also might modify nucleic acids in host cells at sites of inflammation, setting the scene for carcinogenesis or other deleterious changes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Sodium hypochlorite, H₂O₂, organic solvents, and sodium phosphate were obtained from Fisher. Methionine and nuclease P₁ were from Calbiochem (San Diego, CA). bis-(Trimethylsilyl)trifluoroacetamide (BSTFA)¹ + 1% trimethylchlorosilane, N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide + 1% tert-butyl-dimethylchlorosilane (MtBSTFA + 1% tert-butyl-dimethylchlorosilane), silylation grade pyridine, and acetonitrile were from Regis Technologies, Inc. (Morton Grove, IL). Porcine eosinophil peroxidase was provided by Dr. M. L. McCormick (Department of Medicine, University of Iowa). Unless otherwise indicated, all other materials were purchased from Sigma.

Methods

Isolation of Myeloperoxidase (Donor: Hydrogen Peroxide, Oxidoreductase, EC 1.11.1.7)-- HL-60 cells were used as starting material for preparation of myeloperoxidase. The enzyme was isolated by sequential lectin affinity and size exclusion chromatographies (20, 47). Purified myeloperoxidase (A₄₃₀/A₂₈₀ ratio of 0.6) was dialyzed against water and stored in 50% glycerol at 20 °C. Enzyme concentration was determined spectrophotometrically (₄₃₀ = 178 mM¹ cm¹) (48).

Human Neutrophils-- Neutrophils were prepared by density gradient centrifugation and suspended in Hanks' balanced salt solution, pH 7.0 (magnesium-, calcium-, phenol red-, and bicarbonate-free; Life Technologies, Inc.) supplemented with 100 µM diethylenetriaminepentaacetic acid (DTPA) (49). The cells (96% neutrophils; 4% eosinophils) were incubated at 37 °C for 60 min and maintained in suspension with intermittent inversion. The reaction was terminated by addition of methionine to 6 mM and centrifugation of the cells at 400 × g for 10 min. The supernatant was concentrated to dryness under vacuum, dissolved in 0.3 ml of water, centrifuged at 14,000 × g for 10 min, and subjected to HPLC fractionation.

Preparation of Chloride-free Sodium Hypochlorite-- Chloride-free sodium hypochlorite (NaOCl) was prepared by a modification of previously described methods (12). Reagent NaOCl (100 ml) mixed with ethyl acetate (100 ml) was protonated by dropwise addition of concentrated phosphoric acid (final pH  6) with intermittent shaking. The organic phase containing HOCl was washed twice with H₂O, and HOCl was re-extracted into H₂O by the dropwise addition of NaOH (final pH  9). Residual ethyl acetate in the aqueous solution of chloride-free NaOCl was removed by bubbling with N₂. The concentration of NaOCl was determined spectrophotometrically (₂₉₂ = 350 M¹ cm¹; Ref. 50).

Oxidation of 2'-Deoxycytidine, RNA, and DNA-- All reactions were performed in gas-tight vials and initiated by addition with a gas-tight syringe of oxidant (H₂O₂ or HOCl) through a septum while vortexing the sample. Reactions were terminated by addition of a molar excess of L-methionine (6 mM) to eliminate any remaining HOCl, Cl₂, and chloramines. The concentration of H₂O₂ was determined spectrophotometrically (₂₄₀ = 43.6 M¹ cm¹; Ref. 51). The pH dependence of 5-chloro-2'-deoxycytidine formation was determined using reaction mixtures containing phosphoric acid, monobasic sodium phosphate, and dibasic sodium phosphate (final concentration, 50 mM) that had been treated with Chelex-100 (Bio-Rad). The pH of the reaction mixture (without L-methionine) was determined at the end of incubation. RNA and DNA were extracted with one volume of 25:24:1 phenol/chloroform/isoamyl acetate (v/v/v) and one volume of 24:1 chloroform/isoamyl alcohol (v/v) prior to precipitation with [1/2] volume of 7.5 M ammonium acetate and 2 volumes of ice-cold ethanol (52). Reagent N-chloro-2'-deoxycytidine was synthesized (14) by adding 1 mM HOCl to 1 mM 2'-deoxycytidine in chloride-free 50 mM sodium phosphate (pH 7). The reaction mixture was not quenched with methionine. Following a 30-min incubation at 37 °C, N-chloro-2'-deoxycytidine was isolated by HPLC as described below.

Reverse Phase HPLC of 2'-Deoxycytidine Chlorination Products-- 2'-Deoxycytidine oxidation products were analyzed by reverse phase HPLC at a flow rate of 1 ml/min using a C₁₈ column (µPorasil; 5-µm resin, 4.6 × 250 mm; Beckman) with monitoring of absorbance at the 2'-deoxycytidine maximum of 280 nm. Solvent A was 0.1% trifluoroacetic acid (pH 2.5), and solvent B was 0.1% trifluoroacetic acid in methanol (pH 2.5). The column was equilibrated with 95% solvent A. Compounds were eluted with a discontinuous gradient of solvent B. The gradient was 5% solvent B for 4 min, 5-100% solvent B over 20 min, and then 100% solvent B for 10 min. UV-visible spectra for individual HPLC peaks were obtained using a Beckman diode array detector. For mass spectrometric analysis, HPLC fractions were collected and concentrated under vacuum. 5-Chloro-2'-deoxycytidine yield was quantified by comparison of integrated peak areas to standard curves generated using authentic material. For NMR analysis, 10-fold concentrated reaction mixtures were fractionated on a semi-preparative C₁₈ column (µPorasil; 5-µm resin, 10 × 250 mm; Beckman) at a flow rate of 2.5 ml/min with an isocratic gradient consisting of 90% 20 mM ammonium formate (pH 6.3) and 10% methanol. N-Chloro-2'-deoxycytidine (retention time, 10 min) was isolated with a µPorasil C₁₈ column using 5% methanol at a flow rate of 1 ml/min.

NMR Studies-- Reaction products were isolated by HPLC, solubilized in D₂O and analyzed at 25 °C with a Varian Unity-Plus 500 spectrometer (499.843 MHz for ¹H) equipped with a Nalorac indirect detection probe. ¹H chemical shifts were referenced to external sodium 3-(trimethylsilyl)propionate-2,2,3,3-d₄ in D₂O. Spectra were recorded from 8 transients with a 12-s preacquisition delay over a spectral width of 8000 Hz.

Bacterial Culture-- E. coli (American Type Tissue Collection, strain 11775) from frozen stocks was cultured overnight at 37 °C in 5 ml of Luria-Bertani broth (Difco Laboratories, Detriot, MI). and used to inoculate 50 ml of broth. Cells were harvested midway through log phase as assessed by absorbance at 600 nm, then chilled to 4 °C, and washed twice with ice-cold 50 mM sodium phosphate buffer (pH 7). To expose E. coli to the myeloperoxidase-H₂O₂-Cl system, cells (5 × 10⁸ cells/ml) were suspended in buffer B (100 mM NaCl, 50 mM sodium phosphate, pH 4.5) at 37 °C. Following the addition of 50 nM myeloperoxidase and 300 µM H₂O₂, the mixture was incubated for 30 min at 37 °C. The reaction was terminated by adding 6 mM L-methionine and extracting nucleic acids.

Isolation and Hydrolysis of Nucleic Acids-- RNA and DNA were extracted using a modification of the procedure described by Ausubel et al. (53). Bacterial cell walls were digested with 0.4 mg/ml lysozyme for 15 min on ice. Cell lysis was induced by treatment with 1% SDS and 0.1 mg/ml proteinase K for 90 min at 37 °C. Cell lysates were extracted twice with phenol/chloroform/isoamyl acetate (25:24:1 v/v/v) and once with chloroform/isoamyl alcohol (24:1 v/v). Nucleic acids were precipitated with 0.5 volume of 7.5 M ammonium acetate and 2 volumes of ice-cold ethanol. For RNA analysis DNA was removed by DNase I digestion in the presence of placental ribonuclease inhibitor, whereas for DNA analysis RNA was removed with a combination of RNase T₁ and RNase A (52). Nucleases and cell debris were extracted with phenol/chloroform, and nucleic acid polymers were isolated by ethanol precipitation.

Gas Chromatography-Mass Spectrometry (GC/MS)-- For GC/MS analysis, ethanol-precipitated nucleic acids were suspended in 99% formic acid and hydrolyzed for 45 min at 140 °C under a helium or argon atmosphere. For HPLC fractions of cell media, 3 pmol 5-fluoro-2'-deoxycytidine internal standard was added to each sample. Uniform derivatization efficiency between samples was confirmed by monitoring the m/z 300 ion [M^+·  C₄H₉^·] of derivatized 5-fluorocytosine. After drying under vacuum, residual water was removed from the samples by forming an azeotrope with 50 µl of pyridine and again drying the suspension under vacuum. DNA bases were converted to dimethyl, tert-butylsilyl (DMTBS) derivatives with excess N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide containing 1% t-butyl-dimethylchlorosilane (MtBSTFA + 1% tert-butyl-dimethylchlorosilane) in acetonitrile (3:1 v/v) at 100 °C for 60 min. 1-µl aliquots of the reaction solution were analyzed on a Varian Star 3400 CX gas chromatograph equipped with a 12-m DB-1 capillary column (0.2-mm inner diameter, 0.33-µm film thickness; J&W Scientific) interfaced with a Finnigan SSQ 7000 mass spectrometer operated in the positive electron ionization mode. Injector and interface temperatures were 250 and 280 °C, respectively. The initial GC oven temperature was 80 °C for 2 min, followed by a 60 °C/min increasing ramp to 180 °C and a final 10 °C/min ramp to 220 °C. Samples were analyzed in order of increasing concentration, and derivatizing reagent injections were analyzed between samples to ensure that traces of analyte were not adhering to the injector. GC retention times were established for each analysis session by injection of authentic compounds.

Electrospray Ionization-Mass Spectrometry-- HPLC fractions were subjected to solid phase extraction on a C₁₈ column (Supelclean LC-18 SPE tubes, 3 ml; Supelco, Inc., Bellefonte, PA) equilibrated with H₂O to remove TFA. The column was washed with 2 ml of H₂O and eluted with 3 ml of 50% methanol, and the recovered product was dried under vacuum. Full mass scanning, zoom scanning, and low energy collisionally activated dissociation were carried out on a Finnigan LCQ. A 5-µl portion of sample was injected into the electrospray source at a flow rate of 3 µl/min. The electrospray needle was held at 4500 V, and the counterelectrode was held at ground potential. Methanol/water/acetic acid (50:49:1 v/v/v) was used to dissolve samples and as carrier solvent. Helium was used as a damping gas and collision activation partner. The flow of gas (1 ml/min) into the mass analyzer cavity was regulated by a pressure regulator and a capillary restrictor. Flow rates were matched to adjust the partial pressure of helium in the mass analyzer cavity to approximately 10³ Torr. The temperature of the heated capillary was 200 °C. The collision energy was varied by changing the resonance excitation RF voltage. In the full scan mode (m/z 150 to m/z 300), injection time and microscans were 300 ms and 3 s for acquiring each scan, respectively. For each full scan mass spectrum, 10 scans were signal averaged, and the background from the same number of scans was subtracted.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Myeloperoxidase System Oxidizes 2'-Deoxycytidine to 5-Chloro-2'-deoxycytidine-- In preliminary experiments, we exposed adenine, cytosine, guanine, and thymine 2'-deoxynucleosides to the myeloperoxidase-H₂O₂-Cl system in buffer A (100 mM NaCl, 100 µM DTPA, 50 mM sodium phosphate, pH 4.5). After terminating the reaction with methionine (which scavenges HOCl, chloramines, and H₂O₂), we analyzed the reaction mixture by HPLC, monitoring absorbance at 254 nm for purines and 280 nm for pyrimidines. 2'-Deoxycytidine generated the highest yield of product (Fig. 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Reverse phase HPLC chromatogram of 2'-deoxycytidine oxidized by myeloperoxidase. 2'-Deoxycytidine (1 mM) was incubated with (+MPO/H2O2) or without (MPO/H2O2) myeloperoxidase (20 nM) and H2O2 (500 µM) in buffer A (50 mM sodium phosphate, 100 mM sodium chloride, 100 µM DTPA, pH 4.5) for 60 min at 37 °C. Reactions were initiated by the addition of H2O2 and terminated with 6 mM L-methionine. Where indicated, authentic 5-chloro-2'-deoxycytidine was analyzed alone (5-Cl-dC) or added to the complete myeloperoxidase reaction mixture (+MPO/H2O2 + 5-Cl-dC). Inset, normalized UV-visible absorption spectra of 2'-deoxycytidine (dC) and the 2'-deoxycytidine oxidation product generated by myeloperoxidase (5-Cl-dC) (retention time, 12.1 min).

The oxidation product generated by myeloperoxidase eluted from a reverse phase column at a higher methanol concentration than 2'-deoxycytidine; it also absorbed ultraviolet light at a longer wavelength (Fig. 1, inset). The product comigrated with authentic 5-chloro-2'-deoxycytidine (Fig. 1). Moreover, its absorption spectrum and that of authentic 5-chloro-2'-deoxycytidine were indistinguishable. When reagent HOCl was substituted for the enzymatic system, we obtained a product from 2'-deoxycytidine whose chromatographic behavior was identical to that of 5-chloro-2'-deoxycytidine. These observations suggest that the HOCl generated by myeloperoxidase converts 2'-deoxycytidine into 5-chloro-2'-deoxycytidine.

To further investigate the oxidation product structure, we isolated the compounds by HPLC and subjected them to electrospray ionization tandem mass spectrometry. The positive ion mass spectra of the myeloperoxidase product (Fig. 2A), the hypochlorite reaction product, and authentic 5-chloro-2'-deoxycytidine yielded the same major [M + H]⁺ ion at m/z 262. All three compounds also exhibited a prominent ion at m/z 264. The relative abundances of the ions at m/z 262 and 264 reflected that of the natural isotopic abundance of ³⁵Cl and ³⁷Cl, strongly suggesting that the 2'-deoxycytidine oxidation product was monochlorinated. The collisionally activated dissociation tandem mass spectrum of the m/z 262 ion generated a product ion at m/z 146, which is consistent with cleavage of the N-glycoside bond of 5-chloro-2'-deoxycytidine to yield ³⁵Cl-substituted cytosine (Fig. 2B). The collisionally activated dissociation tandem mass spectrum of the m/z 264 ion likewise generated a product ion at m/z 148, consistent with cleavage of the chlorinated nucleoside to yield ³⁷Cl-substituted cytosine (Fig. 2C). The electrospray ionization MS/MS spectrum of the myeloperoxidase product indicates that the chlorine substitution site is likely to reside on the cytosine base of 2'-deoxycytidine.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Positive ion electrospray ionization tandem mass spectrum of the 2'-deoxycytidine oxidation product generated by myeloperoxidase. 2'-Deoxycytidine was oxidized with the complete myeloperoxidase system as described in the legend to Fig. 1, and the major reaction product was isolated by HPLC. A, zoom scan MS analysis of the oxidation product. B and C, MS/MS product ion analysis of the precursor ion at m/z 262 (B) and m/z 264 (C) corresponding to the 35Cl and 37Cl isotopomers of chlorinated 2'-deoxycytidine.

Further structural characterization of the modified nucleobase generated from 2'-deoxycytidine by myeloperoxidase was achieved using GC/MS to obtain an informative electron ionization mass spectrum and GC retention time from material isolated by HPLC. This procedure yields information only about the nucleobase because the N-glycoside bond of the nucleoside is hydrolyzed during the derivatization reactions. After establishing retention times and mass spectra for the trimethylsilyl and DMTBS derivatives of authentic 5-chloro-2'-deoxycytidine, we analyzed the oxidation product generated by either HOCl or the complete myeloperoxidase-H₂O₂-Cl system. The positive ion mass spectra and GC retention times of the trimethylsilyl derivative of the myeloperoxidase reaction product and the HOCl reaction product were essentially identical to those obtained using authentic 5-chloro-2'- deoxycytidine.

The mass spectrum of the myeloperoxidase reaction product exhibited a low abundance ion at m/z 289, consistent with the molecular ion [M^+·] of the bis-trimethylsilyl derivative of 5-chlorocytosine. Trimethylsilyl derivatives typically fragment with loss of CH₃^·; the expected ion was observed at m/z 274. The ions at m/z 274 and m/z 276, as well as those at m/z 289 and m/z 291, exhibited the 3:1 isotopic ratio expected for a monochlorinated compound containing ³⁵Cl or ³⁷Cl. Ions also were observed at m/z 254 [M  Cl]⁺ and m/z 238 ([M  CH₃^·  HCl] or [M  CH₄  Cl]⁺). As anticipated for compounds that lack chlorine, the latter two ions failed to exhibit prominent [M + 2] ions.

We also subjected the DMTBS derivatives of the myeloperoxidase reaction product and the HOCl reaction product to electron ionization GC/MS. Again, the GC retention times and mass spectra of these compounds were essentially identical to those of the DMTBS derivative of authentic 5-chloro-2'-deoxycytidine. The positive ion mass spectra of the DMTBS derivatives were dominated by ions at m/z 316 [M^+·  C₄H₉^·] for ³⁵Cl and 318 [M^+·  C₄H₉^·] for ³⁷Cl. Ions from the DMTBS derivative were concentrated into this single major informative ion, making DMTBS derivatization the method of choice for sensitive analysis.

To confirm that the chlorination site of 2'-deoxycytidine is the 5-position carbon on the nucleobase ring, we oxidized the nucleoside with the complete myeloperoxidase system or HOCl, isolated the product by reverse phase HPLC, and subjected the purified material to ¹H NMR analysis. The spectra obtained from the myeloperoxidase product, the hypochlorous acid product, and authentic 5-chloro-2'-deoxycytidine were essentially identical. Significant features of the product spectra included the lack of a proton resonance at C-5, a downfield shift in the proton resonance at C-6 and conversion of the C-6 proton resonance from a doublet to a singlet, both of which are consistent with substitution of a chlorine atom at the C-5 position (Fig. 3).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   1H NMR spectra of 2'-deoxycytidine (top) and 5-chloro-2'-deoxycytidine generated by the myeloperoxidase system (bottom). The myeloperoxidase (MPO) product was HPLC-purified from the reaction mixture and resuspended in D2O for NMR analysis. The downfield region of the spectrum containing the nucleobase resonances is depicted. Note that the myeloperoxidase oxidation product demonstrates loss of the C-5 proton (peak a), a downfield shift of the C-6 proton (peak b), and conversion of the C-6 proton resonance from a doublet to a singlet peak. All these changes are consistent with substitution of chlorine for the C-5 proton. The triplet signal near 6.3-6.8 ppm is the anomeric proton from the furanose ring of deoxyribose. Protons not observed because of exchange with solvent are designated (L).

Reaction Requirements for Chlorination of 2'-Deoxycytidine by Myeloperoxidase-- We used reverse phase HPLC to characterize the chlorination of 2'-deoxycytidine by myeloperoxidase. Generation of 5-chloro-2'-deoxycytidine required myeloperoxidase, Cl, and H₂O₂; it was blocked by catalase, a scavenger of H₂O₂ (Table I). The overall yield of the reaction was 15% relative to H₂O₂. Two heme enzyme inhibitors, cyanide and azide, also blocked product formation. Chlorination also was inhibited by methionine, which scavenges HOCl, chloramines, and H₂O₂. These results demonstrate that chlorination of 2'-deoxycytidine by myeloperoxidase requires active enzyme, Cl, and H₂O₂. Eosinophil peroxidase, but not lactoperoxidase and horseradish peroxidase, was also capable of chlorinating 2'-deoxycytidine, although with much less efficiency than myeloperoxidase (Table I). Unlike lactoperoxidase and horseradish peroxidase, eosinophil peroxidase can generate low levels of HOCl from H₂O₂ and Cl (54). This property might account for its chlorinating ability.

View this table: [in this window] [in a new window]   Table I Requirements for the conversion of 2'-deoxycytidine into 5-chloro-2'-deoxycytidine by the myeloperoxidase-H2O2-Cl system The complete peroxidase system consisted of 1 mM 2'-deoxycytidine, 20 nM peroxidase, and 300 µM H2O2 in buffer A (100 mM NaCl, 100 µM DTPA, 50 mM phosphate, pH 4.5). The reaction mixture was incubated at 37 °C for 60 min. The reaction was initiated by the addition of H2O2 and terminated by the addition 6 mM L-methionine and 140 nM catalase. The concentration of 5-chloro-2'-deoxycytidine was determined by HPLC analysis. Values are the means of duplicate determinations and are representative of the results found in three independent experiments.

Fig. 4 illustrates the effect of varying reaction conditions on 5-chloro-2'-deoxycytidine generation by myeloperoxidase. Chlorination was maximal at plasma concentrations of Cl (100 mM) and under acidic conditions. The reaction was proportional to H₂O₂ concentration up to 400 µM, with little increase in product yield at higher peroxide concentrations. Its progress curve was initially rapid, and then it gradually slowed.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Reaction requirements for 5-chloro-2'-deoxycytidine generation by myeloperoxidase. 2'-Deoxycytidine (1 mM) was modified by incubation for 60 min at 37 °C with myeloperoxidase (20 nM) and H2O2 (300 µM) in buffer A. The reaction was terminated by the addition of 6 mM L-methionine and 80 nM catalase. Where indicated, the H2O2 concentration (A), chloride concentration (B), pH (C), or reaction time (D) was varied. 5-Chloro-2'-deoxycytidine generation was quantified by HPLC analysis. pH values were determined at the end of the incubation prior to L-methionine and catalase addition.

Chlorination of 2'-Deoxycytidine Involves Molecular Chlorine-- HOCl generated by myeloperoxidase is in equilibrium with Cl₂ through a reaction that requires Cl and a proton (Reaction 2). To determine whether HOCl or Cl₂ is the chlorinating species that generates 5-chloro-2'-deoxycytidine, we investigated the Cl and H⁺ dependence of the synthesis of the compound by reagent HOCl. At pH 4.5 and plasma concentrations of Cl, chlorination of 2'-deoxycytidine was directly proportional to the concentration of HOCl in the reaction mixture (Fig. 5A). In the presence of 100 mM Cl, the yield of 5-chloro-2'-deoxycytidine rose with increasing [H⁺] (Fig. 5B). In contrast, increasing [H⁺] had little effect on the yield of 5-chloro-2'-deoxycytidine in the absence of Cl. Chlorination of 2'-deoxycytidine by HOCl required millimolar concentrations of Cl at pH 4.5 (Fig. 5C). The acidic pH optimum and Cl dependence of chlorination implicates a Cl₂-like species, not HOCl itself, as the oxidizing intermediate in 5-chloro-2'-deoxycytidine generation.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Reaction requirements for 5-chloro-2'-deoxycytidine generation by reagent HOCl. 2'-Deoxycytidine (1 mM) was exposed for 60 min at 37 °C to chloride-free HOCl (300 µM) in buffer B (50 mM sodium phosphate, 100 mM sodium chloride, pH 4.5). The reaction was terminated by the addition of 6 mM L-methionine. Where indicated, chloride-free HOCl concentration (A), pH in the presence () and absence () of 100 mM NaCl (B), and chloride concentration (C) were varied. pH values were determined at the end of the incubation prior to L-methionine addition.

N-Chloro-2'-deoxycytidine Is Not Required for 5-Chloro-2'-deoxycytidine Generation-- Cl₂ might be derived from the reaction of HOCl with Cl (Reaction 2), which would explain the Cl dependence of chlorination. Alternatively, it might be generated indirectly from the chloramine of 2'-deoxycytidine (14). To distinguish between these two possibilities, we determined how adding Cl before or after HOCl affected the yield of 5-chloro-2'-deoxycytidine (Table II). The order of addition should not affect the yield if Cl₂ is derived from the chloramine, whose production does not require Cl. We found, however, that the yield declined dramatically when Cl was added last. This observation implies that reaction conditions that favor the formation of chloramine actually inhibit 5-chloro-2'-deoxycytidine generation, presumably by competing for HOCl.

To further examine the role of chloramines in 5-chloro-2'-deoxycytidine formation, we determined whether isolated N-chloro-2'-deoxycytidine can generate 5-chloro-2'-deoxycytidine. N-Chloro-2'-deoxycytidine was synthesized (14) and isolated by reverse phase HPLC. Its structure was confirmed in three ways: (i) by UV-visible spectroscopy, which revealed absorbance maxima at 227 and 273 nm, which are similar to previously observed values for N-chlorocytosine (14); (ii) by electrospray ionization tandem mass spectrometry; the collisionally activated tandem mass analysis of the material demonstrated that the molecular ion at m/z 262 was converted to an ion at m/z 146, which is consistent with the loss of deoxyribose, and the collisionally activated tandem mass analysis of the ion at m/z 146 showed that a single major ion formed at m/z 111, which is consistent with the loss of chlorine radical from the precursor ion; this ion was not observed with 5-chloro-2'-deoxycytidine; and (iii) by using methionine to convert the product to 2'-deoxycytidine and detecting 2'-deoxycytidine through UV-visible spectroscopy and HPLC.

At plasma concentrations of Cl (100 mM NaCl), there was no detectable conversion of N-chloro-2'-deoxycytidine to 5-chloro-2'-deoxycytidine at either pH 7 or pH 4.5 (Fig. 6). In contrast, 5-chloro-2'-deoxycytidine was generated under strongly acidic conditions (0.1 M HCl), with a yield of 51%. Collectively, these observations provide strong evidence that, under our experimental conditions, 5-chloro-2'-deoxycytidine generation competes with chloramine formation and that N-chloro-2'-deoxycytidine is not an intermediate in the reaction.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effect of pH on generation of 5-chloro-2'-deoxycytidine from the chloramine of 2'-deoxycytidine. N-Chloro-2'-deoxycytidine (250 µM) was incubated for 60 min at 37 °C in 50 mM sodium phosphate buffer containing 100 mM NaCl at either pH 7 (top), pH 4.5 (middle), or in 100 mM HCl (bottom). Reactions were terminated by addition of 6 mM methionine and analyzed by reverse phase HPLC with UV detection at 293 nm. HPLC chromatograms are depicted with indicated peaks corresponding to 2'-deoxycytidine (dC) and 5-chloro-2'-deoxycytidine (5-Cl-dC).

Myeloperoxidase Produces 5-Chlorocytosine in Double-stranded DNA and RNA-- To determine whether myeloperoxidase can chlorinate cytosine residues in polymeric nucleic acids, we incubated 0.4 mg/ml calf thymus DNA or calf liver RNA in buffer A supplemented with 20 nM myeloperoxidase and 300 µM H₂O₂. After 60 min at 37 °C, the reaction was terminated by adding methionine to a final concentration of 6 mM. The reaction mixture was then subjected to acid hydrolysis, and the free nucleobases were converted to their DMTBS derivatives. Electron ionization GC/MS analysis of derivatized acid hydrolysates obtained from DNA or RNA exposed to the complete myeloperoxidase system produced a peak of material in the total ion chromatogram that eluted with the same GC retention time as the bis-DMTBS derivative of authentic 5-chlorocytosine (Fig. 7). Full scan mass spectra of these materials revealed prominent ions at m/z 316 and 318. The relative abundances of the ions was as expected for a chlorine-containing compound and was essentially identical to that observed for the bis-DMTBS derivative of authentic 5-chlorocytosine (Fig. 7, insets). Both myeloperoxidase and H₂O₂ were required for generation of the compound. These results demonstrate that the myeloperoxidase-H₂O₂-Cl system can chlorinate cytosine bases in polymeric DNA or RNA.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Total ion chromatograms and positive ion mass spectra of the DMTBS derivatives of RNA (A) and DNA (B) exposed to the myeloperoxidase-H2O2-Cl system. Calf liver RNA or calf thymus DNA (0.4 mg/ml) was incubated for 60 min at 37 °C in buffer A containing 20 nM myeloperoxidase and 300 µM H2O2. The reactions were terminated with 6 mM L-methionine. The DNA or RNA precipitated from the reaction mixture was hydrolyzed in formic acid, derivatized, and subjected to electron ionization GC/MS analysis. Insets, enlarged view of the elution time frame for 5-chlorocytosine and full scan mass spectrum of the peak eluting with the retention time of authentic 5-chlorocytosine.

5-Chlorocytosine Is Generated Selectively in the RNA of E. coli Exposed to Myeloperoxidase.-- To determine whether the myeloperoxidase system can chlorinate cell-associated nucleic acids, we suspended E. coli in buffer B (50 mM sodium phosphate, 100 mM sodium chloride, pH 4.5) containing 50 nM myeloperoxidase. We then added H₂O₂ to a final concentration of 300 µM and incubated the suspension for 30 min at 37 °C. The reaction was terminated by adding 6 mM L-methionine. To isolate RNA, total cellular nucleic acid was exposed to DNase, and the polymer that remained was isolated by precipitating it with ice-cold ethanol. We used RNase to isolate DNA in a similar manner. Isolated RNA and DNA then were subjected to acid hydrolysis.

When the acid hydrolysate of the E. coli RNA was derivatized with DMTBS and subjected to electron ionization GC/MS, we detected a peak of material with a GC retention time and mass spectrum identical to those of the bis-DMTBS derivative of 5-chlorocytosine. The compound contained the characteristic ³⁵Cl and ³⁷Cl peaks of the [M^+·  C₄H₉^·] ion of 5-chlorocytosine at m/z 316 and 318, respectively (Fig. 8). This signal was not observed when H₂O₂ and myeloperoxidase were omitted from the incubation medium.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Detection by selected ion monitoring of the DMTBS derivatives of cytosine and 5-chlorocytosine in RNA isolated from E. coli exposed to the myeloperoxidase-H2O2-Cl system. E. coli (5 × 108 cells/ml) suspended in buffer B (pH 4.5) was exposed for 30 min at 37 °C to 50 nM myeloperoxidase and 300 µM H2O2. The reaction was terminated by the addition of 6 mM L-methionine. RNA was isolated from total bacterial nucleic acids and hydrolyzed with formic acid, and the nucleobases were derivatized for electron ionization GC/MS with selected ion monitoring. The retention times of derivatized authentic cytosine (m/z 282) and 5-chlorocytosine (isotopomers at m/z 316 and 318) were 6.31 and 6.54 min, respectively.

Selected ion monitoring and comparison with a standard curve suggested that 2% of the cytosine in RNA had been converted to 5-chlorocytosine in E. coli exposed to the complete myeloperoxidase system. In contrast, 5-chlorocytosine was undetectable in the bacterial DNA samples. The limit of detection for 5-chlorocytosine was <0.4% of DNA cytosine, suggesting that myeloperoxidase generates reactive intermediates that selectively chlorinate RNA within bacterial cells.

Activated Human Neutrophils Employ the Myeloperoxidase-H₂O₂-Cl System to Generate 5-Chloro-2'-deoxycytidine-- To determine whether HOCl generated by human neutrophils might chlorinate nucleic acids, we activated human neutrophils with phorbol myristate acetate in physiological salt solution supplemented with 2'-deoxycytidine. Detectable amounts of 5-chloro-2'-deoxycytidine were observed by GC/MS analysis of the cell-conditioned medium when cells were stimulated with phorbol ester or phorbol ester plus superoxide dismutase. 5-Chloro-2'-deoxycytidine formation required activation of the cells with phorbol ester and was inhibited by catalase and heme poisons, implicating myeloperoxidase and H₂O₂ in the reaction (Fig. 9). Superoxide dismutase enhanced the yield of the reaction, perhaps by increasing H₂O₂ availability or preventing inactivation of myeloperoxidase by superoxide (55).

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Reaction requirements for 5-chloro-2'-deoxycytidine generation by activated human neutrophils. Neutrophils (5 × 106/ml) were stimulated with 200 nM phorbol myristate acetate in Hanks' buffered salt solution (pH 7) containing 1 mM 2'-deoxycytidine and maintained in suspension by intermittent inversion. Where indicated, superoxide dismutase (SOD; 10 µg/ml), catalase (CAT; 10 µg/ml), or sodium azide (Azide; 10 mM) were present, or phorbol ester (PMA) was omitted. At the end of a 60-min incubation at 37 °C, cells were pelleted by centrifugation, and 6 mM methionine was added to the medium. The incubation medium was subjected to HPLC, and isolated material was analyzed by GC/MS with selected ion monitoring of the [M  t-butyl·]+ ion of MtBSTFA-derivatized 5-chloro-2'-deoxycytidine (m/z 316).

	DISCUSSION

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Our results demonstrate that the myeloperoxidase-H₂O₂-Cl system can oxidize nucleobases, particularly 2'-deoxycytidine. Multiple lines of evidence indicate that the major product of 2'-deoxycytidine oxidation is 5-chloro-2'-deoxycytidine. First, the HPLC retention time and absorption spectrum of the single major oxidation product that results from exposing 2'-deoxycytidine to myeloperoxidase was identical to that of authentic 5-chloro-2'-deoxycytidine. Second, the electrospray positive ion mass spectrum and collisionally activated tandem mass spectra of the reaction product that contained ³⁵Cl and ³⁷Cl were indistinguishable from those of authentic 5-chloro-2'-deoxycytidine. Third, the retention times on gas chromatography and the mass spectra of two different derivatives of the oxidation product were essentially identical to those of 5-chloro-2'-deoxycytidine. Fourth, the ¹H NMR spectrum of the reaction product is identical to that of authentic 5-chloro-2'-deoxycytidine. Fifth, the optimal reaction conditions for synthesis of the product were similar to those previously reported for HOCl synthesis. Sixth, scavengers of HOCl inhibited the generation of 5-chloro-2'-deoxycytidine by myeloperoxidase, whereas reagent HOCl could replace the enzymatic system. Collectively, these results indicate that reactive species generated by myeloperoxidase chlorinate 2'-deoxycytidine at the 5-position.

HOCl or its conjugate base, hypochlorite, is generally thought to be the chlorinating intermediate generated by myeloperoxidase. However, HOCl is in equilibrium with Cl₂ through a reaction that requires Cl and H⁺ (Reaction 2). Two lines of evidence strongly suggest that a Cl₂-like species is the chlorinating intermediate in 5-chloro-2'-deoxycytidine production. First, chlorination of 2'-deoxycytidine by reagent HOCl was optimal at acidic pH. Second, HOCl failed to chlorinate 2'-deoxycytidine in the absence of Cl (Scheme 1). These observations implicate a Cl₂-like species in the conversion of 2'-deoxycytidine into 5-chloro-2'-deoxycytidine.

View larger version (9K): [in this window] [in a new window]   Scheme 1.

Patton et al. (14) synthesized halogenated cytosine by the addition of high concentrations of reagent hypohalous acid to cytosine in dilute acid; they proposed that 5-chlorocytosine is generated by Cl₂ when N-chlorocytosine is acidifed (3, 14). To address the relative role of Cl₂ generated directly from HOCl (Reaction 2) versus Cl₂ generated indirectly from chloramine, we first determined whether the yield of 5-chloro-2'-deoxycytidine is affected by the order in which Cl is added. If the Cl₂ that mediates 5-chloro-2'-deoxycytidine formation is derived from chloramine, the product yield should be the same or greater when Cl is added to the reaction mixture last rather than first. However, we observed that the yield declined dramatically when Cl was added last. This implies that reaction conditions that favor chloramine formation actually inhibit 5-chloro-2'-deoxycytidine generation. We then directly examined the ability of N-chloro-2'-deoxycytidine to generate 5-chloro-2'-deoxycytidine, but we detected no product under our standard reaction conditions. In contrast, when the chloramine was subjected to strongly acidic conditions, we obtained a high yield of 5-chloro-2'-deoxycytidine, as previously reported (14). Taken together, these observations provide strong evidence that N-chloro-2'-deoxycytidine is not an intermediate in the reaction under our experimental conditions. Instead, generation of 5-chloro-2'-deoxycytidine from HOCl competes with chloramine formation over a physiologically plausible pH range.

We detected 5-chlorocytosine in the formic acid hydrolysates of calf liver RNA, calf thymus DNA and RNA isolated from E. coli exposed to the myeloperoxidase-H₂O₂-Cl system. These observations indicate that the chlorinating intermediate(s) generated by myeloperoxidase is capable of reacting with biological targets remote from the active site of the enzyme. We were surprised to discover, however, that 5-chlorocytosine appeared only in the RNA and not the DNA of bacteria exposed to myeloperoxidase. This selective halogenation suggests that cytosine bases of RNA are vulnerable to oxidation by the chlorinating intermediate, whereas those of intracellular DNA are not, perhaps because of the structure of DNA, the location in the cell, or the interactions with protective proteins. We were unable to detect 5-chlorocytosine in bacteria exposed to activated neutrophils. However, the limit of detection in this analysis was ~1 in 10⁴ nucleobases, and studies of other DNA oxidation products suggest that the physiologically relevant level will be on the order of 1 in 10⁷-10⁸ nucleobases (56). In future studies it will clearly be important to use more sensitive methods to determine whether phagocytes halogenate nucleotides and polymeric nucleic acids of intact bacterial and mammalian cells.

Experiments with cultured neutrophils demonstrated that when activated by phorbol myristate acetate, these cells could convert extracellular 2'-deoxycytidine to 5-chloro-2'-deoxycytidine. Cellular deoxycytidine chlorination was enhanced by superoxide dismutase but blocked in the presence of heme enzyme inhibitors and catalase, consistent with a role for hydrogen peroxide and myeloperoxidase in the reaction.

One important question is whether the acidic pH optimum for 5-chlorocytosine generation by myeloperoxidase is likely to exist in vivo. It is relevant, therefore, that cultured activated macrophages form phagocytic compartments that achieve a pH of less than 4 (57). Also, tissue hypoxia at sites of inflammation or infection results in acidic conditions that would enhance the generation of Cl₂ by myeloperoxidase (58, 59). It is possible that myeloperoxidase-derived chloramines could generate 5-chlorocytosine when exposed to strongly acidic conditions such as those in phagocyte organelles or gastric lesions. Furthermore, human neutrophils use a Cl₂-like species to generate 3-chlorotyrosine from tyrosine in vitro, and we have shown that 3-chlorotyrosine levels are markedly elevated in human atherosclerosis, a chronic inflammatory condition (8, 30).

Detecting 5-chlorocytosine in vivo would strongly support a role for Cl₂ as a physiologic oxidant generated by activated phagocytes. Moreover, chlorinated pyrimidines such as 5-chlorocytosine are attractive candidates for exploring the role of phagocytes in oxidizing nucleic acids because myeloperoxidase is the only known human enzyme that generates HOCl at plasma halide ion concentrations. One strategy for addressing this issue would be to analyze inflammatory tissues for 5-chloro-2'-deoxycytosine. The mass spectrometric methods we have developed should provide powerful tools for investigating the role of myeloperoxidase in damaging nucleic acids in vivo.

If chlorinated pyrimidines are generated in vivo, they could have profound effects on the cells in which they form and even on neighboring cells (60-64). Thus, cultured mammalian cells take up and phosphorylate 5-chloro-2'-deoxycytidine, which then can be incorporated into genomic DNA. Moreover, cellular enzymes deaminate 5-chloro-2'-deoxycytidine to the thymidine analog, 5-chloro-2'-deoxyuridine (60). Such chlorinated pyrimidines exhibit antiviral activity, alter cellular pyrimidine metabolism, induce sister chromatid exchange, and mutate genes (61-64). These observations raise the possibility that myeloperoxidase-catalyzed halogenation of extracellular or intracellular nucleosides and nucleotides may yield products that can be incorporated into nucleic acids, where they would exert mutagenic and cytotoxic effects. Potential substrates for halogenation include plasma nucleosides and nucleobases, intracellular RNA and DNA, deoxycytidine released by T and B cells, and intracellular pools of cytidine and deoxycytidine nucleotides (65, 66).

It is interesting to note that another halogenated pyrimidine, 5-fluorouracil, is known to be incorporated into RNA (67). Many of the cytotoxic effects of this chemotherapeutic agent are thought to be mediated by RNA, suggesting that oxidative damage to RNA may also be biologically relevant. Oxidatively damaged bases also can be incorporated into DNA by DNA polymerase. Organisms have enzymes that cleanse the nucleotide pool of such potentially dangerous species. The bacterial MutT protein, for example, is a nucleoside-triphosphate pyrophosphohydrolase that destroys a mutagenic form of deoxyguanosine triphosphate, 8-oxo-dGTP (68). Mutations in mutT increase the spontaneous mutation rate in the E. coli genome 100-10,000-fold (69-71).

The suggestion that phagocyte-derived toxins could contribute to carcinogenesis by oxidizing cellular nucleic acids is consistent with the association between chronic inflammation and malignancy. Our demonstration that myeloperoxidase can generate reactive chlorinating and nitrating intermediates suggests that nucleic acids may indeed be a target for damage. Detecting chlorinated pyrimidines at sites of inflammation in vivo would strongly support this hypothesis, with important implications for tissue injury and tumor development at chronically inflamed sites.

	ACKNOWLEDGEMENTS

We thank Dr. A. d'Avignon (Department of Chemistry, Washington University) for assistance with NMR studies and Dr. M. L. McCormick (Department of Medicine, University of Iowa) for providing eosinophil peroxidase. Mass spectrometry experiments were performed at the Washington University School of Medicine Mass Spectrometry Resource.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants AG12293, AG15013, and RR00954 and by funds from the American Heart Association and the Monsanto-Searle/Washington University Biomedical Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by a Biophysics Training Grant from the National Institutes of Health.

To whom correspondence should be addressed: Div. of Atherosclerosis, Nutrition and Lipid Research, Campus Box 8046, 660 South Euclid Ave., St. Louis, MO 63110. Fax: 314-362-0811; E-mail: heinecke@im.wustl.edu.

	ABBREVIATIONS

The abbreviations used are: BSTFA, bis-(trimethylsilyl)trifluoroacetamide; DTPA, diethylenetriaminepentaacetic acid; GC, gas chromatography; MS, mass spectrometry; MtBSTFA, N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide; HPLC, high performance liquid chromatography; DMTBS, dimethyl, tert-butylsilyl.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES