In Vitro Adduct Formation of Phosgene with Albumin and Hemoglobin

The development of procedures for retrospective detection and quantitation of exposure to phosgene, based on adducts to hemoglobin and albumin, ...
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Chem. Res. Toxicol. 2000, 13, 719-726

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In Vitro Adduct Formation of Phosgene with Albumin and Hemoglobin in Human Blood Daan Noort,*,† Albert G. Hulst,‡ Alex Fidder,† Ronald A. van Gurp,† Leo P. A. de Jong,† and Hendrik P. Benschop† Department of Chemical Toxicology and Department of Analysis of Toxic and Explosive Substances, TNO Prins Maurits Laboratory, P.O. Box 45, 2280 AA Rijswijk, The Netherlands Received February 7, 2000

The development of procedures for retrospective detection and quantitation of exposure to phosgene, based on adducts to hemoglobin and albumin, is described. Upon incubation of human blood with [14C]phosgene (0-750 µM), a significant part of radioactivity (0-13%) became associated with globin and albumin. Upon Pronase digestion of globin, one of the adducts was identified as the pentapeptide OdC-(V-L)-S-P-A, representing amino acid residues 1-5 of R-globin, with a hydantoin function between N-terminal valine and leucine. Micro-LC/tandem MS analyses of tryptic as well as V8 protease digests identified one of the adducts to albumin as a urea resulting from intramolecular bridging of lysine residues 195 and 199. The adducted tryptic fragment could be sensitively analyzed by means of micro-LC/tandem MS with multiplereaction monitoring (MRM), enabling the detection in human blood of an in vitro exposure level of g1 µM phosgene.

Introduction Phosgene is an important intermediate for industrial production of insecticides, isocyanates, plastics, aniline dyes, and resins with an estimated production of almost 1 billion pounds per year. As a result of this extensive usage, thousands of industrial workers are potentially at risk of exposure to phosgene. In addition, phosgene can be formed by the thermal decomposition of chlorinated hydrocarbons during fires, and thus be a potential hazard to firefighters. Exposure to phosgene vapor may cause immediate irritation of the eyes, nose, throat, and respiratory tract and increases lipid peroxidation and pulmonary vascular permeability, contributing to noncardiogenic pulmonary edema (1, 2). It is estimated that exposure of humans to >30 ppm‚min causes lung damage, whereas a 10-fold higher dose may lead to fatal lung edema (3). The estimated LCt50 (i.e., product of exposure concentration and time which is lethal for 50% of the exposed population) for phosgene in humans is 800 ppm‚ min (3200 mg‚min‚m-3) for a 2 min exposure. A specific problem in the case of phosgene intoxication is the latency period in the development of lung edema, which is 4-15 h in the case of moderate intoxications. Presumably, acylation of proteins at alveolar-capillary membranes leads to leakage of fluid from the capillary into the interstitial portions of the lung. Reliable diagnosis of exposure to phosgene other than observation of the developing edema by means of chest roentgenology is not available. Consequently, precious time is lost before the severity of the intoxication can be assessed, i.e., the very period of time in which dose* To whom correspondence should be addressed: TNO Prins Maurits Laboratory, P.O. Box 45, 2280 AA Rijswijk, The Netherlands. Phone: +31 15 2843497. Fax: +31 15 2843963. E-mail: [email protected]. † Department of Chemical Toxicology. ‡ Department of Analysis of Toxic and Explosive Substances.

related therapy is supposed to be most effective (4). At present, only passive dosimeters are available for those at risk of accidental exposure to phosgene, which monitor the external dose of phosgene (5). It follows that development of simple, rapid methods of diagnosis and dosimetry of exposure to phosgene is highly worthwile. Our approach toward diagnosis is based on analysis of phosgene adducts with proteins present in the blood, which are presumably formed upon respiratory exposure. It is well-known that phosgene is highly reactive toward amino, hydroxyl, and thiol groups (6), not only in organic solvents but also in aqueous solutions (7). Binding of phosgene, formed from chloroform by metabolic activation, to proteins and endogeneous compounds has already been observed by others. For example, Pohl et al. (8, 9) reported binding of [14C]phosgene formed from [14C]chloroform to microsomal protein. Binding to glutathione has also been observed (10). Recently, binding of phosgene, generated by chloroform metabolism, to phospholipids in liver mitochondria was reported (11). Pereira and Chang (12) observed binding of radioactivity to hemoglobin upon administration of [14C]chloroform to rats. In subsequent investigations (13), they identified the adduct as a reaction product of phosgene with a cysteine moiety of hemoglobin. Evidence that inhaled phosgene can actually enter the bloodstream was presented by Sciuto et al. (14). Upon exposure of rodents to a high dose (Ct ) 1740 mg‚min‚m-3) of phosgene, a change in absorption at 413 nm in the plasma was observed, indicating that the integrity of the erythrocytes had been affected. In view of the long lifetime of hemoglobin in erythrocytes (ca. 120 days in humans), analysis of hemoglobin adducts may aid in monitoring the cumulative dose of exposure of workers to phosgene. Analysis of adducts to albumin may also be worthwile. In view of the short half-life of phosgene in blood, albumin may be a more effective scavenger than hemoglobin, since an

10.1021/tx000022z CCC: $19.00 © 2000 American Chemical Society Published on Web 07/08/2000

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erythrocyte has to be entered before reaction with hemoglobin can occur. In this report, we demonstrate that phosgene binds effectively to hemoglobin and albumin upon in vitro exposure of human blood to phosgene. Complete characterization of two adducts is presented, as well as a mass spectrometric procedure for assessing exposure to phosgene based on an adduct to albumin.

Materials and Methods Caution: Phosgene is a highly toxic agent which should be handled only in fume cupboards by experienced personnel. Materials. Phosgene was used as a 20% solution in toluene and was obtained from Fluka. [14C]Phosgene (specific activity of 53 mCi/mmol, >97% pure, 20% solution in toluene) was purchased from BlyChem (Billingham, U.K.). Trypsin (TPCKtreated, type XIII from bovine pancreas, EC 3.4.21.4), Pronase (protease type XIV from Streptomyces griseus, EC 3.4.24.31), and V8 protease (endoproteinase Glu-C, protease type XVII-B from Staphylococcus aureus strain V8, EC 3.4.21.19) were obtained from Sigma (St. Louis, MO). Human blood was obtained from healthy volunteers in our laboratory, with consent of the donor and approval of the TNO Medical Ethical Committee. Instrumentation. HPLC was performed using a Gilson (Villers-le-Bel, France) HPLC system consisting of a model 305 master pump, a model 306 slave pump, a model 805 manometric module, and a model 811C dynamic mixer, in combination with a Source 15RPC column (Pharmacia, Uppsala, Sweden). The eluent (flow rate of 1 mL/min) was 0.1% trifluoroacetic acid (TFA)1 in H2O with a linear gradient to 0.1% TFA in CH3CN/ H2O (various ratios). The eluate was monitored at 214 nm with a Spectroflow 757 UV detector (Applied Biosystems, Ramsey, NJ) and with a radiochromatography detector (Radiomatic, model Flo-one Beta series A 500, Meriden, CT) with UltimaFlo (Packard, Meriden, CT) as the scintillation cocktail. Liquid scintillation countings were performed with a Minaxi Tri-Carb 4000 series scintillation counter (Packard) with Hionic Picofluor (Packard) as the scintillation cocktail. Micro-LC/tandem MS analyses were performed on a Q-TOF mass spectrometer or on a VG Quattro II triple-quadrupole mass spectrometer (both from Micromass, Manchester, U.K.) which were coupled to an HPLC system consisting of a Waters (Bedford, MA) 2690 HPLC module or an HPLC system consisting of two Waters solvent delivery systems (models 590 and 510) under the control of a solvent programmer (Waters, model M680), respectively. For gradient LC, the milliliters per minute flow rate from the delivery system was converted to a microliters per minute flow rate using an Acurate microflow processor (model IC-400-VR; LC Packings, Zu¨rich, Switzerland). A PepMap C18 column (15 cm × 0.3 mm; LC Packings) was used. Samples were applied through a Valco injection valve (VICI, Schenkon, Switzerland), equipped with a 10 µL sample loop. Conditions for mass spectrometric analyses are given in the relevant entries. 1H and 13C NMR spectra were recorded on a Varian (Palo Alto, CA) VXR 400S spectrometer operating at 400.0 and 100.6 MHz, respectively. Chemical shifts are given in parts per million relative to HDO (1H) or dioxane (13C). Peptides were synthesized on an Abimed (Langenfeld, Germany) AMS 422 peptide synthesizer using commercially available building blocks (Novachem, La¨ufelfingen, Switzerland). Slide-a-Lyzer cassettes (0.1-0.5 mL) were obtained from Pierce (Rockford, IL). Sep-Pak C18 cartridges (model Classic, short body) were obtained from Waters (Milford, MA). Centrex UF-2 (10 kDa molecular mass cutoff) centrifugal ultrafilters were procured from Schleicher & Schuell (Keene, NH). Incubation of Human Blood with Phosgene or [14C]Phosgene and Isolation of Globin and Albumin. To human 1 Abbreviations: CID, collision-induced dissociation; MRM, multiplereaction monitoring; TFA, trifluoroacetic acid.

Noort et al. blood (2 mL) was added a solution of phosgene or [14C]phosgene in CH3CN/toluene [9/1 (v/v), 20 µL]. After incubation (under gentle shaking) for 2 h at 37 °C, plasma and erythrocytes were separated by centrifugation at 3000g for 10 min. Subsequently, globin was isolated according to the method of Bailey et al. (15), and albumin was isolated according to the method of Bechtold et al. (16). Pronase Hydrolysis of Globin, Sample Workup, and Micro-LC/Tandem MS Analysis of OdC-(V-L)-S-P-A. Pronase hydrolysis was performed as decribed previously (17), with the exception that the protein was dissolved in H2O/PBS [1/1 (v/v), 400 µL; brought to pH 8 with aqueous NaOH] instead of 50 mM NH4HCO3 because of the low solubility of globin in the latter. After incubation for 2.5 h at 37 °C, the mixture was filtered through a centrifugal ultrafilter (10 kDa cutoff) with centrifugation at 4000g. Cleanup of the sample using Sep-Pak C18 cartridges was performed as described previously (17), with the exception that 4 mL instead of 2 mL of 0.1% TFA/20% CH3CN was used prior to elution with 0.1% TFA/40% CH3CN. The 40% CH3CN fraction was analyzed by means of micro-LC/ tandem MS on a Q-TOF mass spectrometer. In this case, the pentapeptide OdC-(V-L)-S-P-A was detected by collision-induced dissociation (CID) of a [M + H]+ ion at m/z 512.3. In case of exposure to [14C]phosgene, an analogous compound is detected with a [M + H]+ ion at m/z 514.2. Operation conditions of the mass spectrometer were as follows: scan range, m/z 50-1700; scan time, 2.5 s; cone voltage, 30 V; collision energy, 18 eV; and argon pressure, 10-4 mbar. The injection volume was 10 µL. LC was performed on a PepMap C18 column (15 cm × 0.3 mm). Gradient elution using H2O with 0.2% HCOOH and CH3CN with 0.2% HCOOH as eluents A and B, respectively, was performed. The flow scheme was as follows: 95% eluent A at a flow rate of 0.1-0.5 mL/min from 0 to 5 min and, subsequently, 95% eluent A to 80% eluent B at a flow rate of 0.5 mL/min from 5 to 90 min. Flow rates were reduced by a splitter: from 0 to 5 min, 1 f 5 µL/min, and subsequently 5 µL/min. Synthesis of OdC-(V-L)-S-P-A. To a solution of V-L-S-P-A (9 mg, 18 µmol), obtained by solid phase peptide synthesis, in H2O (5 mL) at pH 9, was added a solution of phosgene in toluene (60 µL, 1.9 M). The reaction mixture was stirred at room temperature, while the pH was kept at 9 by addition of aqueous NaOH (40 mM) with pH-stat equipment. After 0.5 h, an additional portion of phosgene solution (60 µL) was added. The reaction mixture was stirred for an additional 16 h at room temperature at pH 9, after which HPLC analysis showed complete conversion into a compound with a longer retention time. The reaction mixture was neutralized with acetic acid and concentrated to a small volume (1 mL). The crude product was purified by preparative HPLC using a PepRPC 10/10 column. The desired fractions were pooled and lyophilized. Yield: 4.7 mg (white fluffy solid). Electrospray MS: m/z 512.3 [M + H]+. 1H NMR (D O): δ 4.9 (broad m, 2H, serine and leucine CH-R), 2 4.5 (dd, 1H, proline CH-R, J ) 5.2 and 8.7 Hz), 4.4 (q, 1H, alanine CH-R, J ) 7.3 Hz), 4.3 (d, 1H, valine CH-R, J ) 3.5 Hz), 4.0-3.8 (m, 4H, serine CH2-β and proline CH2-δ), 2.3 (m, 7H, valine CH-β, leucine CH2-β, proline CH2-β and CH2-γ), 1.5 (m, 4H, alanine CH3, J ) 7.3 Hz, leucine γ-CH), 1.1-0.9 [4 × d, 12H, 4 × CH3, leucine (J ) 6.7 Hz) and valine (J ) 7.0 Hz)].13C NMR (D2O): δ 177.3, 177.0, 174.5, 172.1, 170.6 (4 × CdO, amide, COOH), 159.2 (CdO, hydantoin), 63.2 (serine β-CH2), 61.1 (proline R-CH, valine R-CH), 54.6 (serine R-CH), 53.0 (leucine R-CH), 49.3 (alanine R-CH), 48.7 (proline δ-CH2), 37.0 (leucine β-CH2), 30.5 (valine β-CH), 29.9 (proline β-CH2), 25.2 (leucine γ-CH, proline γ-CH2), 23.0 and 20.7 (leucine 2 × δ-CH3), 18.7 and 16.7 (valine 2 × γ-CH3), 15.8 (alanine β-CH3). Carboxymethylation and Tryptic Digestion of Albumin. Carboxymethylation with iodoacetic acid and tryptic digestion were performed as described previously (17). Thus, shortly, to a solution of albumin (3 mg) in a buffer (300 µL) containing 6 M guanidine hydrochloride, 100 mM Tris-HCl, and 1 mM EDTA (pH 8.3) was added dithiothreitol (5 mg), and the solution was

Analysis of Phosgene Adducts incubated at 55 °C for 40 min. Subsequently, iodoacetic acid (sodium salt, 10 mg) was added and the mixture incubated at 40 °C for 30 min. The clear solution was transferred into a Slidea-Lyzer cassette (0.1-0.5 mL), and the solution was dialyzed against aqueous 50 mM NH4HCO3 (3 L) for 16 h. Trypsin (2% w/w) was added, and the mixture was incubated at 37 °C for 4 h. Finally, the sample was filtrated through a filter with a 10 kDa cutoff with centrifugation at 4000g, to remove the enzyme. Micro-LC/Tandem MS Analysis of OdC-(T25-T28). The tryptic digests of albumin were analyzed for (A-S-S-A-K*-Q-R)(L-K*-Z-A-S-L-Q-K), with a CdO bridge between K* residues and with Z being carboxymethylcysteine [abbreviated as OdC(T25-T28)] by means of micro-LC/tandem MS analysis. Operation conditions of the Q-TOF mass spectrometer were as follows: cone voltage, 40 V; collision energy, 32 eV; and argon pressure, 10-4 mbar. The injection volume was 10 µL. LC was performed on a PepMap C18 column (15 cm × 0.3 mm). Elution was performed as described above. In case of exposure to phosgene, OdC-(T25-T28) is detected by CID of a [M + 2H]2+ ion at m/z 861.0. In case of exposure to [14C]phosgene, an analogous compound is detected with a [M + 2H]2+ ion at m/z 862.0. For more sensitive detection of OdC-(T25-T28), analyses were performed in the multiple-reaction monitoring (MRM) mode (transitions [M + 2H]2+ m/z 861.0 f 747.5 and [M + 2H]2+ m/z 861.0 f 773.5) on a Quattro II triple-quadrupole mass spectrometer. Operating conditions were as follows: cone voltage, 40 V; collision energy, 30 eV; argon pressure, 5 × 10-3 mbar; and dwell time, 1 s. LC conditions were as described above. Digestion of Albumin with V8 Protease. To a solution of S-carboxymethylated albumin, from human blood which had been exposed to [14C]phosgene (0.75 mM), in aqueous 50 mM NH4HCO3 (100 µL, 6 mg/mL), was added a solution of V8 protease in aqueous 50 mM NH4HCO3 (0.2 mg/mL, 100 µL). The mixture was incubated for 2.5 h at 37 °C. Subsequently, the mixture was analyzed by means of micro-LC/tandem MS. In this case, the modified S192 fragment G-K-A-S-S-A-K*-Q-R-L-K*-ZA-S-L-Q-K-F-G-E, with a 14CdO bridge between K* residues and with Z being carboxymethylcysteine, is detected by CID with a [M + 3H]3+ ion at m/z 741.7. Operation conditions of the mass spectrometer were as follows: cone voltage, 40 V; collision energy, 27 eV; and argon pressure, 10-4 mbar. Further conditions were as described above. Molecular Modeling. The Brookhaven Protein Data Bank (PDB) structure of human serum albumin (18, 19) was imported into QUANTA96 (Molecular Simulations, San Diego, CA); polar hydrogen atoms were added, and the remaining hydrogen atoms were treated implicitly. Lys and Arg residues were protonated, and Asp and Glu residues were deprotonated. The resulting molecule was subjected to energy minimization using the CHARMm method, version 2.3 (20), with a distance-dependent dielectric constant of 4.0. The phosgene adduct was created by inserting a carbonyl group between the side chain nitrogen atoms of Lys195 and Lys199, creating the urea bridge and removing superfluous hydrogen atoms to ensure electrical neutrality. The resulting structure was subsequently refined by CHARMm energy minimization.

Results Quantitation of Binding. For quantitation of phosgene binding to globin and albumin, human blood was incubated with 14C-labeled agent (0.75 mM, 0.075 mM, and 7.5 µM; specific activity of 53 mCi/mmol) for 2 h at 37 °C. After isolation of globin and albumin, the proteins were dissolved in a solution of 6 M guanidine, 100 mM Tris, 1 mM EDTA, and 1 mM DTT, and the amount of 2 Cleavage in 50 mM NH HCO by V8 protease is restricted to the 4 3 carboxylic side of glutamic acid residues; fragments are denoted as S1, S2, etc.

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Figure 1. HPLC analysis (Source 15RPC column) of a Pronase digest of globin isolated from human blood that was treated in vitro with [14C]phosgene (0.75 mM): (a) detection of radioactivity and (b) UV detection (214 nm). The eluent (flow rate of 1 mL/ min) was 0.1% TFA in H2O with a linear gradient to 0.1% TFA in CH3CN/H2O (42/58, v/v) over the course of 20 min. The arrow denotes the peak for Od14C-(V-L)-S-P-A. Table 1. Binding of [14C]Phosgene to Globin and Albumin upon Treatment of Human Blood with Various Concentrations of the Agent [[14C]phosgene] (µM) 7.5 75 750

% of total radioactivity bound to globin

pmol of adduct/mg of globin

not detectable not detectable 2.2 11.8 5.5 295

% of total radioactivity pmol of bound to adduct/mg albumin of albumin 13 12 8.2

23.8 220 1500

radioactivity was determined with liquid scintillation counting. A survey of the results is given in Table 1. Covalent binding was assessed by reversed phase HPLC analysis with UV and radiometric detection of the proteins. Radioactivity coeluted with the UV-positive material. Identification of Globin Adducts. We first directed our attention toward identification of adducted amino acids in hemoglobin. In a first attempt globin, isolated from human blood that had been exposed to [14C]phosgene (0.75 mM), was digested with trypsin. Several radioactive peaks were present in the HPLC chromatogram (results not shown). We were not able to identify any modified peptide in this digest by means of microLC/tandem MS. Upon HPLC analysis of a Pronase digest of the same globin sample, several radioactive peaks were observed (see Figure 1). The two peaks in the late-eluting region of the chromatogram (representing 13 and 15% of the radioactivity bound to globin, respectively) could be isolated by elution of the Pronase digest on a SepPak C18 cartridge. Mass spectrometric analysis of the Sep-Pak eluate revealed the presence of a compound with a [M + H]+ ion at m/z 514.2 (Figure 2a). Upon analysis of a digest from globin isolated from human blood exposed to nonlabeled phosgene (1 mM), a compound with a [M + H]+ ion at m/z 512.3 was present, having an identical retention time and a similar fragmentation pattern (Figure 2b). The identity of this adduct was tentatively determined to be OdC-(V-L-S-P-A), corresponding to amino acids 1-5 of R-globin. The fragmentation pattern of both precursors suggests that modification has occurred in the VL part of the pentapeptide; y2′′ (m/z

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Figure 2. Product ion spectrum of (a) molecular ion [M + H]+ (m/z 514.2) of Od14C-(V-L)-S-P-A in a Pronase digest of globin isolated from human blood that was exposed to [14C]phosgene (0.75 mM) and (b) molecular ion [M + H]+ (m/z 512.3) of OdC(V-L)-S-P-A in a Pronase digest of globin isolated from human blood that was exposed to phosgene (1 mM).

187, PA) and y3′′ (m/z 274, SPA) fragments are present, as well as a2, a3, b3, and b4 fragments whose magnitudes are increased with 26 amu compared to the fragments of the native sequence. For final identification, the adduct was chemically synthesized. Reaction of V-L-S-P-A with phosgene in aqueous solution at pH 9 afforded a single product which coeluted with the first late-eluting peak in Figure 1 (see the arrow). Upon micro-LC/tandem MS analysis, the synthetic compound exhibited an identical tandem MS spectrum, and it coeluted with the modified peptide present in the Pronase digest. To exclude the involvement of the serine hydroxyl function, the synthetic compound was treated with acetic anhydride in pyridine. A mass increase of 42 amu was observed, indicating the presence of a free hydroxyl function. The identity of the compound was firmly established by NMR spectroscopy of the synthetic adduct. In the 13C NMR spectrum, a strong signal at 159 ppm was observed which was not present in the 13C NMR spectrum of the native peptide. 1H NMR (COSY) analysis showed the presence of NH signals of valine, alanine, and serine. The leucine NH signal was absent, and in addition, the R-CH function of leucine did not couple with an NH function, indicating the absence of the leucine NH function. From these results, we conclude that modification has occurred between valine and leucine under formation of a hydantoin function (see the inset in Figure 2b). The identity of the other radioactive peaks in Figure 1 could not be unraveled. It was investigated whether the hydantoin is a suitable biomarker for retrospective detection of exposure to phosgene. Unfortunately, determination of exposure levels of 0.1 mM. Lower exposure levels could not be assessed since blank samples contained significant levels of a compound with an identical retention time and a similar tandem MS spectrum. This compound probably results from N-terminal carboxylated R-globin, which is formed by reaction of R-globin with CO2 and of which significant levels are present in human blood (25). The hydantoin function between the valine and leucine residues might form either spontaneously from the carboxy-amino hemoglobin or during isolation or processing of globin. To test this hypothesis, we exposed an aqueous solution of synthetic V-L-S-P-A to gaseous CO2 at pH 7.4 and room temperature. The expected N-carboxylated peptide was formed together with trace amounts of a compound which coeluted with the pentapeptide hydantoin and gave a similar mass spectrum. This interference can probably also occur by reaction of R-globin, as present in human hemoglobin, with CO2 dissolved in blood. From these results, it is clear that the specific hemoglobin adduct is not suitable as a sensitive biomarker for exposure to phosgene.

Analysis of Phosgene Adducts

We did not succeed in identifying any modified amino acids or peptides in Pronase digests of albumin by microLC/tandem MS; mainly rapidly eluting material resulted after Pronase digestion. Consequently, modified peptides are present in the bulk of amino acids and peptides. Several discrete radioactive peaks resulted upon tryptic digestion of albumin from human blood exposed to [14C]phosgene and subsequent HPLC analysis with radiometric detection. The urea peptide resulting from intramolecular bridging of Lys195 and Lys199 could be fully characterized by micro-LC/tandem MS analyses of tryptic as well as V8 protease digests. It was tentatively determined that 22% of radioactivity associated with albumin was bound to Lys195 and Lys199. Modification of these particular lysine residues has been observed by other workers. Walker (26) reported the specific modification of Lys199 by acetylsalicylic acid. Ding et al. (27, 28) and Zia-Amirhosseini et al. (29) reported that Lys195 and Lys199 were major sites for covalent binding of acyl glucuronides to human serum albumin. Yvon et al. (30, 31) showed that benzyl penicilloyl groups, the allergenic metabolites of penicillin, bind inter alia to the Lys195 and Lys199 residues of human serum albumin. The reactivity of the Lys199 residue is due to its relatively low pKa value, which is attributed to the His242 residue in proximity (32). Our molecular model corroborates this hypothesis, by placing the imidazole ring of His242 within 3.5 Å of the Lys199 nitrogen atom. The model also shows residues Glu292 and Tyr150 in proximity to Lys199, presumably further activating the nitrogen atom. Reaction of the two lysines with phosgene, under formation of a urea derivative, is expected to introduce extra strain in the albumin molecule. However, the protein backbone and the surrounding residues appear to be unaffected by the chemical modification. Apparently, the strain is relieved by the movement of flexible lysine side chains, according to our adduct model. The adducted tryptic fragment could be sensitively analyzed by means of micro-LC/tandem MS with MRM, enabling the detection in human blood of an in vitro exposure level of 1 µM phosgene. Further enhancement of the minimum detectable concentration was hampered by small signals in the blank samples at the same retention time as the analyte. It cannot be excluded that CO2 (or HCO3-), which is abundantly present in the bloodstream, gives rise to the formation of the same albumin adduct, as was also the case for the globin adduct. However, in contrast to the assay for analysis of the globin adduct described above, we feel that the assay for analysis of the tryptic peptide adduct from albumin might be useful for monitoring actual exposures to phosgene. For example, in the case of a single exposure to 0.1 × LCt50 (320 mg‚min‚m-3) of phosgene, a concentration of 1.3 µM can be reached in blood, assuming that only 10% of the total dose will be absorbed in the blood (respiratory rate of 20 L/min, total blood volume of 5 L). This will probably give adduct levels which can still be detected with our assay. Chronic exposure to lower levels of phosgene might also be able to be detected by using the assay for analysis of the albumin adduct, since albumin adducts are in general quite stable and will accumulate in time. In conclusion, we demonstrated that phosgene binds to hemoglobin and albumin after exposure of human blood to the agent in vitro. Two adducts were identified, and the development of methods for their mass spectro-

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metric analysis was initiated. We believe the results presented here constitute a promising basis for further development of procedures which enable diagnosis of internal exposure to phosgene. In future work, we will attempt to optimize the analysis of the modified T25T28 tryptic fragment from albumin by means of improved sample cleanup. The use of the identified adducts as haptens for development of an immunoassay for rapid detection of exposure to phosgene can also be envisaged.

Acknowledgment. This work was supported by the Directorate of Military Medical Services of the Ministry of Defence, The Netherlands. We are grateful to Dr. B. L. M. van Baar for stimulating discussions, to Mrs. L. F. Chau for performing the solid phase peptide syntheses, and to Mr. S. van Krimpen for performing the NMR analyses.

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