94
Chem. Res. Toxicol. 1991, 4 , 94-101
Reactions of Aqueous Chlorine in Vitro in Stomach Fluid from the Rat: Chlorination of Tyrosine Michael G . Nickelsen,? Anthony Nweke,? Frank E. Scully, Jr.,*gt and H. Paul Ringhandt Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529-0126, and Health Effects Research Laboratory, U S . Environmental Protection Agency, 26 West St. Clair, Cincinnati, Ohio 45268 Received August 2, 1990
Aqueous chlorine reacts with tyrosine to form ring-chlorinated products. Ring substitution occurs at Cktyrosine mole ratios greater than 1. Because the nitrogen function of amides is much less reactive than that of amines, the aromatic ring of N-acetyltyrosine is chlorinated a t chlorinesubstrate mole ratios less than 1. When an aqueous solution of the gastric protein pepsin was chlorinated (37 “C, 45 min), tyrosine residues were chlorinated a t pH 2 but not at pH 8. The carbohydrate, protein, and chloride concentrations in stomach fluid from fasted rats were determined. When varying concentrations of aqueous chlorine (20-180 mg/L C12) were added to the stomach fluid a t pH 2, tyrosine residues were mono- and dichlorinated on the aromatic ring. The amount of mono- to dichlorination products varied with the concentration of aqueous chlorine. A mechanism is proposed. The implications for toxicological studies involving chlorinated drinking water are discussed.
Introduction It has been estimated that on a daily basis the average person drinks 1-2 liters of water containing a residual disinfectant. In the United States the most commonly used disinfectant is aqueous chlorine. In a survey of 80 localities 58% of the drinking waters tested contained residual aqueous chlorine concentrations between 0.4 and 2.8 mg/L (Cl,) (1). Recently, the American Water Works Association recommended a goal of a “minimum free chlorine residual of 0.5 mg/L ... maintained generally throughout the distribution system” (2). There have been few reports of toxicological studies of the effects of drinking aqueous chlorine. Abdel-Rahman et al. (3)have shown that blood glutathione levels decrease significantly in Sprague-Dawley rats within 30 min after oral administration of aqueous chlorine solutions of 10 and 40 mg/L (1.4 X and 5.6 X M). Meier et al. ( 4 ) have found that oral administration of aqueous chlorine (pH 8.5) at levels of 4 and 8 mg/kg body weight per day for 5 weeks induced sperm-head abnormalities in B6C3F1 mice. Revis et al. (5) added aqueous chlorine at concentrations of 2 and 15 mg/L at either pH 6.5 or pH 8.5 to the drinking water of pigeons for three months. They claimed significant increases in serum thyroid T, and T4 levels at very low doses of disinfectant. In a recent more extensive study this group found no dose-dependent effects of aqueous chlorine on plasma T, levels and very few groups that differed from controls (6). Because aqueous chlorine is a potent oxidizing and chlorinating agent, it is not likely that it is directly responsible for any of these effects. Most probably any health effects associated with the ingestion of aqueous chlorine are due to the byproducts of its reactions with organic and inorganic components of saliva and stomach fluid. For several years we have studied the reactions of aqueous chlorine in stomach fluid as part of an effort to evaluate the potential health effects of *Author to whom correspondence should be addressed. Old Dominion University. U.S. Environmental Protection Agency. 0893-228~/91/2704-0094$02.50/0
drinking chlorinated water (7-10). In this paper we wish to report that free tyrosine molecules present in stomach fluid, as well as tyrosine residues bound in gastric proteins, react with aqueous chlorine at remarkably low concentrations to product chlorinated byproducts.
Materials and Methods General Procedures. All reagents were obtained from commercial sources and were of the highest purity available. Amino acid standards, pepsin (porcine), and N-acetyltyrosine were obtained from Sigma Chemicals, Inc. [3BC1]Chloridewas obtained as its sodium salt in neutral solution from ICN Biomedicals, Inc., with a specific activity of 13.78 or 16 mCi/g of C1-. Standard solutions of tyrosine and N-acetyltyrosine were prepared in chlorine demand free (CDF) water. CDF water was prepared from high-purity water (Milli-Q water system, Millipore Corp.) which had been chlorinated to 5 mg/L, allowed to stand 1 h, brought to a boil, cooled, and irradiated in a glass container overnight with ultraviolet (UV) light (Photronix Corp., Medway, MA) to remove residual chlorine. Concentrated solutions of aqueous chlorine (approximately 14 mM) were prepared by dilution of commercially available solutions of sodium hypochlorite (Clorox) and were standardized by iodimetric titration as previously described ( I I). Concentrations of residual chlorine were determined by the DPD ferrous titrimetric method (Method 408 D) (12). The propionic acid used in acid hydrolysis was distilled prior to mixing with 6 N HCl. All amino acids were analyzed by high-performance liquid chromatography [HPLC, described previously ( I I ) ] after precolumn derivatization with o-phthalaldehyde (OPA) (13). The two-solvent gradient program used to chromatograph OPA derivatives of amino acids utilized 10% acetonitrile/gO% water (containing 1%acetic acid, adjusted to pH 4 with concentrated NaOH) as solvent A. Solvent B consisted of 10% water (1% acetic acid, pH 4)/90% acetonitrile. The program began with a linear solvent gradient (1.5 mL/min) from 100% A to 95% A/5% B over 5 min. After a 10-min isocratic elution the gradient was continued over 25 min to 75% A/25% B. A 5-min isocratic elution was then followed by a 15-min linear gradient to 65% A/35% B, and this was followed by a final linear gradient over 5 min to 10% A/90% B. Amino acids were identified by comparing the retention times of their OPA derivatives with those of standards. A one-point 0 1991 American Chemical Society
Chlorination of Tyrosine in Stomach Fluid calibration with a standard sample of amino acids was used to approximate concentrations. Solutions of N-acetylamino acids, pepsin, and stomach fluid were hydrolyzed with equal volumes of acid (70% 6 N HC1, 30% propionic acid) for 30 min at 155 "C (heating block temperature) in sealed hydrolysis tubes (Pierce Chemical Co. catalogue no. 29560). Gas chromatography/mass spectroscopy (GC/MS) was performed on a Varian 3400 GC coupled to a Finnigan Incos 50 mass spectrometer and data system operated in a purged splitless mode. A 0.32 mm i.d. X 30 m DB-5 fused silica capillary GC column with a 0.25-pm film thickness (J&W Scientific) was used for both test mixtures and chlorinated stomach fluid samples. After a splitless injection for 1.0 min purge was applied. GC/MS conditions used were as follows: electron impact ionization, 70 eV; GC injector temperature, 240 "C; column temperature program, 50-280 "C at 4 "C/min; column flow rate (helium), 1.8 mL/min. The instrument was tuned with decatluorotriphenylphosphine(DFTPP) (14). The mass spectrometer was scanned from 35 to 650 amu every 0.85 s. Characterization of Rat Stomach Fluid. Rat stomach fluid was obtained by the method described previously (8). The stomach fluid of several animals was pooled to give a sufficient working volume. The stomach fluid was then centrifuged and passed through a 0.45-pm glass fiber filter to remove particulates. Three separate composite samples of stomach fluid were used. The characteristics of the three samples are listed in Table I. The total protein concentration of the stomach fluid was determined by the Biuret method (1.9, the total carbohydrate concentration of the stomach fluid was determined by reaction with a-naphthol and sulfuric acid (16),and the total chloride ion concentration was determined by using a Scientific Products chloride-specific electrode (type H3728-204). The electrode was calibrated according to manufacturer's instructions with standard solutions of NaCl ranging in concentration from to lo-' M. The reference electrode used was a Corning Model 476029 silver/silver chloride electrode. Chlorination of Tyrosine. The pH of a solution of aqueous chlorine (4.8 X M) was adjusted to 10 with sulfuric acid. Ten(lo-) mL aliquots of a solution of tyrosine (2.5 x M) were diluted in a 100-mL volumetric flask to approximately 80 mL with CDF water which had been adjusted to pH 10 with 1M sodium hydroxide. The solutions were chlorinated with 0, 0.5, 1, 2, or 4 equiv (ch1orine:nitrogen) and diluted to the mark with CDF water. Two-milliter portions of each solution were incubated in 3-mL amber vials with constant stirring at 37 "C for 45 min. A 50-pL aliquot of the product mixture was derivatized with an equal volume of OPA derivatizing solution containing 6mercaptoethanol (131, and 10 pL was analyzed by HPLC. Standards for identification of the chlorinated tyrosines were prepared by chlorination of a solution of tyrosine with 2 or 3 equiv of aqueous chlorine. A blank (deionized water) was also chlorinated and derivatized in a similar manner. The ethyl acetate solutions of the derivatized samples were analyzed by gas chromatography/ mass spectroscopy (GC/ MS). Chlorination of N-Acetyltyrosine. The pH of CDF water used as diluent and the pH of a solution of aqueous chlorine were adjusted to the desired working pH (pH 2 or 8) with either sulfuric acid or sodium hydroxide. Ten- (lo-) mL aliquots of 2.5 X loV3 M N-acetyltyrosine were diluted to approximately 80 mL with CDF water. Solutions were chlorinated with 0, 0.5, 1, 2, and 4 equiv (ch1orine:nitrogen) of aqueous chlorine (5.1 X M) and diluted to the mark with CDF water. Two-milliter portions of each solution were incubated in 3-mL amber vials with constant stirring at 37 "C for 45 min. After incubation the solutions were dechlorinated for 30 min with an amount of sulfite equivalent to the amount of chlorine used. The product mixtures (200 pL) were acid hydrolyzed with an equal volume of acid (70% 6 N HCl, 30% propionic acid) for 30 min a t 155 OC (heating block temperature) in sealed hydrolysis tubes (Pierce Chemical Co. catalogue no. 29560). The pH of each hydrolysate was adjusted to 10 with 125 pL of 50% aqueous sodium hydroxide before being derivatized with OPA derivatizing solution (23) and analyzed (50 pL) by HPLC. Chlorination of N-Acetyltyrosine with [3BCl]Hypochlorite. Aqueous chlorine and CDF water were adjusted to pH 8.0. A 1.0-mL aliquot of an aqueous solution of N-acetyltyrosine (2.5
Chem. Res. Toxicol., Vol. 4, No. I, 1991 95
x M) was mixed with 80 pL of an aqueous solution of N a y 1 (13.78 pCi/0.0278 mmol of C1,0.035 pCi/pL). The solution was chlorinated with 2 equiv of aqueous chlorine (6.5 X lo-' M) and diluted to 10 mL with CDF water. In this manner [3BCl]hypochlorite (>75% isotopically pure %C10-) was produced by rapid isotope exchange between HOC1 and [3sCl]chloride (17). The solution was incubated (37 "C, 45 min) and dechlorinated with an equivalent of sodium sulfite (30 pL of a 0.167 M solution). The product mixture (200 pL) was hydrolyzed with an equal volume of acid (70% 6 N HCl, 30% propionic acid) for 30 min at 155 "C (heating block temperature) in sealed hydrolysis tubes (Pierce Chemical Co. catalogue no. 29560). The pH of the hydrolysate was adjusted to 10 with 50% aqueous sodium hydroxide before it was derivatized with OPA derivatizing solution (13)and a 1WpL aliquot analyzed by HPLC. Seventy (70) 1.50-mL fractions were collected and analyzed by liquid scintillation counting (LSC) as described previously (8). Counting efficiency for 36Clwas 96%. Chlorination of Pepsin. A solution of pepsin was prepared by dissolving 1.00 g in 1.00 L of CDF water and adjusting the pH of the solution to 8.0 with 1M NaOH or to 2.0 with sulfuric acid. Working solutions of aqueous chlorine were prepared by diluting a stock solution to 40,200, and 750 mg/L (0.56,2.8, and 10.6 mM C12)and adjusting their pH to 8.0 or to 2.0. Equal volumes (0.500 mL) of pepsin solution and aqueous chlorine were mixed thoroughly so that solutions were effectively chlorinated to 20,100, and 350 mg/L (0.28,1.4, and 5.3 mM C12)at the desired pH. The solutions were incubated in amber vials with constant stirring a t 37 "C for 45 min. After incubation the solutions were dechlorinated for 30 min with an amount of 0.167 M sodium sulfite equivalent to the amount of chlorine used. A 200-pL aliquot of the product mixture was hydrolyzed with an equal volume of acid (70% 6 N HC1,30% propionic acid) for 30 min at 155 "C (heating block temperature) in sealed hydrolysis tubes (Pierce Chemical Co. catalogue no. 29560). The pH of the hydrolysate was adjusted to 10 with 50% aqueous NaOH. A 60-pL aliquot of this mixture was derivatized with an equal volume of OPA derivatizing solution (13),and a 100-pL aliquot was analyzed by HPLC with fluorescence detection. Chlorination of Pepsin with H o w l . To each of two 250-pL aliquots of pepsin solution (1.00 g/L, pH 8.0 or 2.0) was added 19 pL of commercial [3BCl]chloridesolution (16 mCi/g of Cl-, 3.41 x g of Cl-IpL). Working solutions of aqueous chlorine were prepared by diluting a stock solution to 100 and 200 mg/L (1.4 and 2.8 mM C12) and adjusting their pH to 8.0 or to 2.0. Equal volumes (250 pL) of pepsin solution and aqueous chlorine were mixed thoroughly so that solutions were effectively chlorinated to 50 and 100 mg/L (0.70 and 1.4 mM C12, respectively) at the desired pH. The resulting solutions were incubated (37 "C, 45 min), dechlorinated (10 pL of 0.167 N sulfite for 30 min), and hydrolyzed. A 60-pL aliquot of each mixture was derivatized with an equal volume of OPA derivatizing solution (13)and a 150-pL aliquot of this analyzed by HPLC. Seventy (70) 1.50-mL fractions were collected from each chromatogram and analyzed by LSC. Chlorination of Rat Stomach Fluid. The pH values of aliquota of rat stomach fluid and of a solution of aqueous chlorine were adjusted to 2.0 with sulfuric acid. Appropriate amounts of a stock solution of aqueous chlorine (1250 mg/L C12, 17.6 mM) were diluted with CDF water (adjusted to pH 2 with sulfuric acid) to provide working solutions of 100,200, and 400 mg/L (C12, 1.4, 2.8, and 5.6 mM). Aliquots of rat stomach fluid (200 pL) were mixed with equal volumes of these working solutions so that the fluid was effectively chlorinated with 50,100, and 200 mg/L C12 (0.70, 1.4, and 2.8 mM, respectively). The solutions were thoroughly mixed and incubated in an amber vial with constant stirring (37 "C, 45 min). After incubation the solutions were dechlorinated for 30 min with an amount of sulfite (0.167 M) equivalent to the chlorine dose used. A 200-pL aliquot of each reaction mixture was hydrolyzed with an equal volume of acid (HCl/propionic acid). The hydrolysate was adjusted to pH 10 with 50% aqueous NaOH. A 200-pL aliquot was derivatized with 100 p L of OPA derivatizing solution, and a 200 pL aliquot was analyzed by HPLC with fluorescence detection. Chlorination of Rat Stomach Fluid with HOWL A 1.0-mL aliquot of the rat stomach fluid used in the previous experiment was ultrafiltered in the following manner to remove chloride. The
96 Chem. Res. Toxicol., Vol. 4 , No. 1, 1991
Nickelsen et al.
Table I. Chemical Characteristics of Composite Samples of Stomach Fluid from the Fasted Rat total volume carbohydrate rat stomach no. of fasting recovered, protein concn, chloride concn,mM fluid sample animals time, h mL pH concn, mg/Ln mg/Lb 1 5 48 11.0 4.27 ndc ndc ndc 2 6 24 16.5 4.78 1215 14.5 16.0 3 8 24 23.0 4.63 2835 35.0 35.2 Protein concentration in mg/L as bovine serum albumin (BSA). *Carbohydrateconcentration in mg/L as sucrose. mined. sample was placed in an Amicon Model 12 ultrafiltration chamber fitted with an Amicon YC05 filter (nominal molecular weight cutoff 500) and diluted to 10 mL with CDF water. The filtration chamber was pressurized with nitrogen to 26 psi until the volume was reduced to 1.0 mL. This procedure was repeated twice. The filtered fluid was stored at -20 "C until it was used. To 1.00 mL of the ultrafiltered fluid was added 0.100 mL of [36Cl]chloride(174 pCi/mL, 17.4 pCi, 13.8 mCi/g of C1) to give M, replacing the a final chloride concentration of 3.5 X chloride that had been removed by ultrafiltration. Appropriate amounts of a stock solution of aqueous chlorine (1250 mg/L, 17.6 mM C12) were diluted with CDF water (adjusted to pH 2 with sulfuric acid) to provide working solutions of 40,80,100,130, and 170 mg/L (0.56, 1.12, 1.4, 1.8, and 2.4 mM Clz). Aliquots of rat stomach fluid (200 pL) were mixed with equal volumes of these working solutions so that the fluid was effectively chlorinated to 20,40,50,65, and 85 mg/L C12 (0.28,0.56,0.70,0.92,and 1.2 mM, respectively). The isotopic purity of the hypochlorous acid present was greater than 98% H036Cl. The solutions were thoroughly mixed and incubated in an amber vial with constant stirring (37 "C, 45 min). The solutions were dechlorinated for 30 min with an amount of sulfite (0.167 M) equivalent to the chlorine dose used. A 200-pLaliquot of each reaction mixture was hydrolyzed with an equal volume of acid (HCl/propionic acid). The hydrolysate was adjusted to pH 10 with 50% aqueous NaOH. A 200-pL aliquot was derivatized with 100 pL of OPA derivatizing solution, and a 200-pL aliquot was analyzed by HPLC with fluorescence detection. Seventy (70) 1.50-mL fractions were collected and analyzed by LSC. Identification of Chlorinated Tyrosine Residues i n Chlorinated Rat Stomach Fluid by Gas Chromatography/ Mass Spectroscopy. The pH of two aliquots of rat stomach fluid (400 pL) was adjusted to 2 with sulfuric acid. The aliquots were chlorinated with 200 p L of HOC1 to final chlorine concentrations of 80 and 180 mg/L (1.1and 2.5 mM C12). After incubation at 37 "C for 45 min in an amber vial, the fluid was cooled, dechlorinated with 1 equiv of sodium sulfate (20 or 45 pL of a 3.38 X M solution), and acid hydrolyzed with an equal volume of acid (HCl/propionic acid). The hydrolysate was lyophilized to remove all acid and water. The N,O-bis(heptafluorobutyry1)tyrosine n-propyl esters were prepared by the method of Burleson et al. (18)using heptafluorobutyric anhydride (HFBA) obtained from Pierce Chemical Co. (Rockford, IL). A blank (deionized water) was also chlorinated and derivatized in a similar manner. The ethyl acetate solutions of the derivatized samples were analyzed by gas chromatography/mass spectroscopy (GC/MS).
Results Characterization of Rat Stomach Fluid. The protein, carbohydrate, a n d chloride ion concentrations of t h e three separate composite samples of rat stomach fluid used throughout these studies as well as t h e pH values of t h e fluid a s it was recovered are listed in Table I.
Chlorination of Tyrosine and N-Acetyltyrosine. W h e n tyrosine was chlorinated with 1 equiv of aqueous chlorine (pH 10) HPLC analysis of t h e o-phthalaldehyde (OPA) derivatives revealed t h a t 66% of t h e tyrosine had reacted after 30 min at 37 "C. T h e r e were no OPA derivatives of new products observed. However, when t h e ch1orine:nitrogen ratio was increased t o greater t h a n one, two new OPA derivatives were noted. By contrast, when N-acetyltyrosine was chlorinated with 1equiv of aqueous chlorine, dechlorinated, hydrolyzed, a n d derivatized with
= not deter-
HOC1
+ N- Acetyltyrosine
HOC1 : N-Ac-Tyr
p H 8.0
4 : 1
h
2 /h
I
20
'
I
'
LO
I
I
60
Retention Time l m i n l
Figure 1. HPLC chromatograms of OPA-derivatizable products obtained after dechlorinationand acid hydrolysis of the products of the reaction of N-acetyltyrosine (N-Ac-Tyr) with varying amounts of aqueous chlorine (pH 8). HOC1:N-Ac-Tyr molar ratios were 0:1,0.5:1, 1:1, 2:1, and 4:l.
OPA, HPLC analysis revealed that 92% of the tyrosine had reacted, and two new OPA derivatives were observed (Figure 1)with retention times (43 and 48 min) identical with those of compounds formed on chlorination of tyrosine a t t h e higher chlorine concentrations. GC/MS analysis (described below) of chlorination products of tyrosine identified mono- and dichlorinated ring substitution products. Since t h e OPA derivative of the chlorination product with a retention time of 43 min was formed at t h e lower chlorination levels, it was assumed t o be t h e monochlorinated tyrosine. When N-acetyltyrosine was chlorinated with 2 equiv of aqueous chlorine (pH 8) containing W1-labeled hypochlorite, t h e OPA derivatives of t h e hydrolysis products with retention times of 43 a n d 48 min revealed incorporation of 36Cl atoms i n a ratio of 1.1t o 1, respectively. Assuming t h e products of t h e chlorination reaction are t h e N-acetyl derivatives of chlorotyrosine and dichlorotyrosine, respectively, t h e two chlorination products were formed in a mole ratio of 2.2 t o 1.0, respectively. Chlorination of Tyrosine Residues in Pepsin. An aliquot of a n aqueous solution of pepsin (1000 mg/L) was acid hydrolyzed a n d analyzed by HPLC for amino acids
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 97
Chlorination of Tyrosine i n Stomach Fluid HOC1
+
500
PEPS\N p H 2
t
1
100 ppm
I I I
4
3
200
a 150 100 50
o j
NO CI2
I
I
0
,
,
,
,
,
,
,
, LO
20
,
,
, 60
FRACTION NUMBER
Figure 3. Radiochromatogram of the OPA-derivatized product mixture after dechlorination and acid hydrolpis from the reaction (45 min, 37 O C ) of HOT1 (65 mg/L as Cl,, pH 2) with ultrafiltered stomach fluid.
I
10
'
I
20
'
1
30
'
1
LO
'
/
50
'
,
60
'
,
70
RETENTION TIME l m i n l
Figure 2. HPLC chromatogram of the OPA-derivatized hydrolyzable amino acids found in pepsin after reaction for 45 min at 37 "C with HOCl (pH 2). Concentrationsof HOCl used were 0 and 100 mg/L (as Cl,). The retention time of tyrosine is indicated by the arrow and the dashed line. after precolumn derivatization with OPA. The bottom chromatogram in Figure 2 was obtained. Fourteen (14) of the 20 essential amino acids were clearly identifiable in the sample at varying concentrations. When pepsin was chlorinated at pH 2 with various concentrations of aqueous chlorine, dechlorinated, hydrolyzed, derivatized with OPA, and chromatographed, the reaction of tyrosine residues could be observed. At a chlorine concentration of 50 mg/L (0.7 mM) approximately 75% of the tyrosine had reacted. The upper HPLC chromatogram shown in Figure 2 was obtained with an aqueous chlorine concentration of 100 mg/L (Cl,, 1.4 mM). The OPA derivatives of the major amino acids eluted between 14 and 65 min. The OPA derivative of tyrosine eluted at 37 min. When pepsin was mixed with 50 mg/L [36C1]hypochlorous acid (as CI,, 0.7 mM) at pH 2, incubated for 45 min at 37 "C, dechlorinated, hydrolyzed, derivatized with OPA, and analyzed by HPLC followed by subsequent analysis of fractions with liquid scintillation counting (LSC), two %C1incorporation products were formed which had retention times the same as the products of the chlorination of tyrosine and N-acetyltyrosine (43 and 48 min). The relative amount of 36Clradioactivity incorporated in each peak was 1.0 to 1.2, respectively. Assuming the products are chlorotyrosine and dichlorotyrosine, respectively, the two chlorination products were formed in a mole ratio of 1.65 to 1.0, respectively. In a control experiment pepsin was incubated (45 min at 37 "C) with [W]chloride at pH 2. Sulfite was added and the protein was hydrolyzed, derivatized with OPA, and analyzed by HPLC followed by subsequent analysis of fractions with liquid scintillation counting (LSC). No radioactive fractions were observed other than those containing [36Cl]chloride,which eluted in the void volume of the column.
When pepsin was chlorinated at pH 8, the number of tyrosine residues that reacted was proportional to the chlorine concentration used, but no W1-chlorinated tyrosines were formed when pepsin was reacted with [%Cl]hypochlorite. Chlorination of Tyrosine Residues in Stomach Fluid. An aliquot of a pooled sample of stomach fluid recovered from eight animals was analyzed after acid hydrolysis for amino acids by precolumn derivatization with OPA followed by HPLC. Fourteen (14) of the 20 essential amino acids were identified in the sample at varying concentrations. When stomach fluid was reacted with aqueous chlorine at concentrations above 20 mg/L (as Cl,, 0.28 mM) at pH 2, dechlorinated, hydrolyzed, derivatized with OPA, and chromatographed,tyrosine residues were observed to react. When rat stomach fluid (sample 3, Table I), ultrafiitered to remove chloride and reconstituted with [36C1]chloride, was chlorinated with H0%1 to 65 mg/L (as Cl,, 0.92 mM) at pH 2, dechlorinated, hydrolyzed, derivatized with OPA, and chromatographed, and fractions were analyzed by liquid scintillation counting, the reconstructed radiochromatogram shown in Figure 3 was obtained. The successful design of this experiment required that unlabeled chloride be removed from stomach fluid and replaced with [36Cl]chloride,since H036Cl would undergo rapid isotope exchange (17) with normal unlabeled chloride in stomach fluid and the labeled chlorinating agent would be lost before it could react with other substrates. The large radioactive peak eluting between 3 and 8 min is due to [36Cl]chloride,which elutes in the void volume of the column. The radioactive peaks of interest eluted at 43 and 48 min. The retention times of these peaks suggest that tyrosine residues in stomach fluid formed 3sC1-labeledring substitution products similar to those formed in the reactions of tyrosine and N-acetyltyrosine with aqueous chlorine. The products are presumed to be 3-chlorotyrosine and 3,5-dichlorotyrosine, as discussed below. Assuming this to be the case, the amounts of radioactivity incorporated can be used to determine the relative amounts of tyrosine reacted and the products formed at each concentration of aqueous chlorine used. Results are plotted in Figure 4. When stomach fluid was reacted with aqueous chlorine under similar conditions, but at pH 8, no %C1incorporation products were observed. GC/MS Identification of Tyrosine Chlorination Products. Chlorinated tyrosine standards and chlorinated/hydrolyzed samples of stomach fluid were deriva-
98 Chem. Res. Toxicol., Vol. 4, No. I , 1991
Nickelsen et al.
Table 11. Major Ions for 70-eV Mass Spectra of Chlorinated Tyrosines Formed on Chlorination of Tyrosine and Rat Stomach Fluid
chlorinated/derivatized samole
mlz (relative intensitv)
.
LOO
100
-1.
350
2 0
7
4
4
-
.
300j 250
K
i >
150
0 ' 0
I
1
I
T
T
1
I
I
10
20
30
LO
50
60
70
80
O
CHLORINE DOSE I m g I L I A
TYROSINE
L MONOCHLOROTYROSINE
DlCHLOROTYROSlNE
Figure 4. Relative amounts of unchlorinated tyrosine, monochlorinated tyrosine, and dichlorinated tyrosine present in the stomach fluid from the rat which had been chlorinated with ~ 0 3 ~ 1 .
tized with heptafluorobutyric anhydride (HFBA) and converted to their n-propyl esters by the method of Burleson et al. (18). Electron impact mass fragmentation patterns of the HFBA n-propyl esters of the chlorinated tyrosines have been studied previously (18). The 70-eV mass spectra of these compounds do not contain parent ions. The major fragment ions (base peaks) in the literature spectra of both the N,O-bis(heptafluorobutyry1)chlorotyrosine n-propyl ester (M+ = 649) and the N,Obis(heptafluorobutyry1)dichlorotyrosine n-propyl ester (M+ = 683) are due to loss of heptafluorobutyramide (C3F,CONHz, M+ - 213) by a @-eliminationfollowed by a McLafferty rearrangement of the ester with loss of propene (19). The chlorinated tyrosines were identified in the mixtures obtained in the present study by selected ion monitoring of the base peaks reported by Burleson et al. (18).
Table I1 contains a list of the major ions used in structural elucidation and all other ions with intensities greater than 5% for 70-eV mass spectra of N,O-bis(heptafluorobutyryl) n-propyl esters of chlorinated tyrosine standards obtained in this study. Also included in the table are derivatives of compounds isolated from chlorinated stomach fluid which have GC retention times corresponding to the chlorinated tyrosine derivatives. The mass spectrum of the monochlorinated tyrosine derivative isolated from chlorinated stomach fluid closely matched both the standard and the published spectrum (18) of the compound. The base peak (m/z 394) was the same as that reported previously (18). Abundances of major fragments
at m / z 562 ( l l % , due to a-cleavage of the n-propyl ester function), m / z 337 (27%, due to benzylic cleavage), m / z 436 [22% due to loss of heptafluorobutyramide, C3F,CONH, (M+- 213), by a @-elimination],m/z 377 (12%), and m / z 349 (6%) correspond very closely with literature values (18). The spectrum of the N,O-bis(heptafluorobutyr1)dichlorotyrosine n-propyl ester standard is very similar to the spectrum of the compound isolated from chlorinatedlderivatized stomach fluid with corresponding retention time. The relative intensities of ions with m/z greater than 169 are very similar in both the spectra reported here and the published spectrum. However, the base peak reported by Burleson et al. (18) is m / z 428 while the base peak in both the spectrum of the standard and that of the stomach fluid sample reported here is m/z 69. Ions due to fragmentations similar to those observed in the spectrum of the monochlorotyrosine derivative described above are present. The major differences occur in the intensities of ions associated with the fragmentation of the heptafluorobutyryl groups: m/z 169 (C3F7+),m/z 131 (C3F5+), m / z 119 (C2F5+),and m / z 69 (CF3+)(20). Related ions and the presumed structure of their fragments are as follows: m/z 150 (C3F6*+)and m/z 100 (CzF4*+,the perfluorinated vinyl cation radical). A second attempt to obtain a spectrum of a standard sample of a dichlorinated tyrosine derivative only confirmed the results obtained earlier. Differences in the results obtained here and published results can only be attributed to differences in the tuning of the two instruments. The total ion chromatogram and the m/z 428 selected ion chromatogram of the derivative identified in stomach fluid are shown in Figure 5.
Discussion Kinetic Considerations. The second-order rate constants for the reaction of amino nitrogen compounds with aqueous chlorine to form organic N-chloramines are very high. The rate constant for the reaction of glycine, for example, is 1.1X ld M-' s-'(21). However, the mechanism for the chlorination of amino nitrogen requires the reaction of a free amino group with hypochlorous acid as illustrated for the reaction of glycine: HZNCHZCOO- + HOCl
-
ClNHCHzC00-
+ HzO
(1)
Consequently, the rate is dependent on the fraction of free available chlorine that is present as HOCl at a given pH and the fraction of free amino groups present at that pH. The rate is fastest at pH values between 7.5 (equal to the
Chem. Res. Tonicol., Vol. 4, No. 1, 1991 99
Chlorination of Tyrosine in Stomach Fluid
z c W
'"1
J,
500
TOTAL ION CURRENT
1500
1000
2000
2500
SCAN NUMBER
Figure 5. Total ion current chromatogram and the selected ion chromatogram ( m / z 428) for GC/MS analysis of rat stomach fluid which had been chlorinated to 180 mg/L for 30 min (37 "C). dechlorinated with sulfite, esterified with HCl/n-propyl alcohol, and derivatized with heptafluorobutyric anhydride (HFBA).
pK, value of HOC1) and approximately 10 (the pK, of the amino nitrogen of the amino acid). Because the fraction of free amino groups decreases as the pH decreases, the rate decreases by a factor of 10 for every pH unit below 7 for a given set of concentrations. Lee (22)and Soper and Smith (23) studied the rate of reaction of phenols with aqueous chlorine at pH 5-12. At pH 8 the observed rate constant is 6.15 X lo3 M-' min-'. This is comparatively slow compared with the reactions of amino nitrogen compounds at that pH. When tyrosine was reacted at pH 8 with less than 1 equiv of chlorine, no products which were OPA-derivatizable were observed, because the reaction of the amino nitrogen was much faster than reaction of the side-chain aromatic group. Any N-chlorotyrosine remaining after 30 min of reaction is converted back to its parent amine during derivatization in the presence of P-mercaptoethanol. Any tyrosine reaction products formed would result from deamination of the amino group (24-29). Because amide nitrogens are considerably less reactive than amino groups (3O),chlorination of the aromatic side chain is the predominant reaction of N-acetyltyrosine at ch1orine:nitrogen mole ratios
