Bicarbonate inhibits N-nitrosation in oxygenated nitric oxide solutions.

N-Nitrosation in oxygenated nitric oxide (NO middle dot) solutions was previously shown to be significantly inhibited by phosphate and chloride presumably by anion scavenging of the nitrosating agent nitrous anhydride, N2O3 (Lewis, R. S., Tannenbaum, S. R., and Deen, W. M. (1995) J. Am. Chem. Soc. 117, 3933-3939). Here, bicarbonate is shown to exhibit this same inhibitory effect. Rate constants for reaction of morpholine, phosphate, and bicarbonate with N2O3 relative to N2O3 hydrolysis at pH 8.9 were determined to be (3.7 +/- 0.2) x 10(4) M-1, (4.0 +/- 0.9) x 10(2) M-1, and (9.3 +/- 1.5) x 10(2) M-1, respectively. The morpholine and phosphate rate constants at pH 8.9 are similar to those reported at pH 7.4 assuring that these results are relevant to physiological conditions. The rate constant for this previously unrecognized reaction of bicarbonate with N2O3 suggests the strong scavenging ability of bicarbonate; accordingly, bicarbonate may contribute to reducing deleterious effects of N2O3. This is biologically important due to substantial bicarbonate concentrations in vivo, approximately 30 mM. Bicarbonate was previously shown to alter peroxynitrite reactivity; however, carbon dioxide is the probable reactive species. Bicarbonate is therefore potentially important in determining the fate of two reactive species generated from nitric oxide, N2O3 and ONOO-, and may thus act as a regulator of NO middle dot-induced toxicity.

Nitric oxide (NO ⅐ ) is an important physiological messenger that is produced by several different cell types and is involved in many processes in vivo including inhibition of platelet aggregation, blood vessel relaxation, and neurotransmission (8). An alternative to these physiologically important pathways is the formation of reactive species that may ultimately result in cytotoxic or mutagenic events by a number of possible mechanisms. Mutagenic effects may arise from the reaction of nitric oxide with superoxide (O 2 . ) forming peroxynitrite (ONOO Ϫ ) that can in turn oxidize many types of molecules including DNA. Alternatively, reaction of NO ⅐ with molecular oxygen results in the formation of nitrous anhydride (N 2 O 3 ) which can cause cytotoxic effects through the nitrosation of both primary and secondary amines. DNA bases containing primary amine functionalities undergo nitrosative deamination upon treatment with NO ⅐ resulting in a modified base (9,10). N 2 O 3 can also nitrosate secondary amines forming carcinogenic N-nitro-samines that can damage DNA following metabolic activation. N 2 O 3 can modify other cell constituents including protein sulfhydryl groups and low molecular weight thiols such as glutathione resulting in S-nitrosothiols. The kinetics of morpholine N-nitrosation by nitric oxide at physiological pH have recently been studied by Lewis et al. (1) using a novel reactor that allows continuous and simultaneous measurements of NO ⅐ , nitrite (NO 2 Ϫ ), and N-nitrosomorpholine (NMor) 1 concentrations. In this system, N 2 O 3 was identified as the key nitrosating agent (1). The measured rate constant for the reaction of morpholine with N 2 O 3 relative to N 2 O 3 hydrolysis was 4.0 ϫ 10 4 M Ϫ1 . A key finding was the inhibitory effect of phosphate and chloride on morpholine nitrosation; the rate constants for reaction of these anions with N 2 O 3 relative to N 2 O 3 hydrolysis were 4.0 ϫ 10 2 M Ϫ1 and 9.0 ϫ 10 1 M Ϫ1 , respectively. Participating anions react with N 2 O 3 forming nitrosyl compounds (XNO) that can in turn react with amines or be hydrolyzed to HNO 2 and ultimately nitrite. At physiological pH, hydrolysis of XNO is much faster than nitrosation of amines by XNO; therefore, the anions will scavenge N 2 O 3 and lower the rate of N-nitrosation (1). Other anions including nitrate, nitrite, thiocyanate, and perchlorate have little or no effect on nitrosation.
During the course of DNA deamination studies, 2 it was found that NO ⅐ -related deamination of calf thymus DNA at physiological pH was inhibited by sodium bicarbonate, NaHCO 3 . Bicarbonate therefore seems to protect biomolecules from the nitrosative effects of NO ⅐ presumably due to an ability to scavenge N 2 O 3 and consequently inhibit nitrosation at pH 7.4. The initial evidence for bicarbonate's inhibitory effect at pH 7.4 prompted the use of a modified reactor similar to that developed by Lewis and Deen (11) to determine the rate constant for reaction of bicarbonate with N 2 O 3 . However, bicarbonate cannot be studied reliably at pH 7.4 in this system due to extensive argon degassing resulting in a shift in the equilibrium between bicarbonate and carbon dioxide and a consequent pH increase. The rate constants were therefore determined at pH 8.9.
As shown here, bicarbonate is important in determining the fate of N 2 O 3 , the product of nitric oxide oxidation; in addition, bicarbonate has been reported to alter the rate of reactions of peroxynitrite, the product of nitric oxide reaction with superoxide (2,3,12). However, it has now been demonstrated that CO 2 is the actual species that reacts with peroxynitrite (5-7). The reactivity of bicarbonate/CO 2 with both N 2 O 3 and ONOO Ϫ and the relatively high concentrations of bicarbonate in interstitial and intracellular fluids (up to 30 mM) suggest that bicarbonate is a key determinant of the fate of the reactive species generated from nitric oxide and that bicarbonate may * This work was supported by National Institutes of Health Grants CA09112, CA26731, ES04675, and ES07020. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ To whom correspondence should be addressed: Massachusetts Institute of Technology, Room 16-822A, Cambridge, MA 02139. Tel.: 617-253-3729; Fax: 617-252-1787; E-mail:srt@mitvma.mit.edu. be protective of NO ⅐ -induced toxicity. Consequently, the presence of bicarbonate must be taken into account in all experiments with nitric oxide both in the presence and absence of reactive oxygen species.

MATERIALS AND METHODS
Reagents-Morpholine (Aldrich Chemical Co.) was used for the Nnitrosation studies. Phosphate buffer (0.01 M) at pH 7.4 was prepared with K 2 HPO 4 and KH 2 PO 4 using double-distilled water. Solutions containing 0.04 M bicarbonate were prepared fresh by adding sodium bicarbonate to the 0.01 M phosphate buffer. Nitric oxide (Matheson, Gloucester, MA) was passed through a column of 4 -8 mesh soda lime to remove NO x impurities. Argon (Ar), after passage through an oxygen trap, was mixed with NO ⅐ using electronically controlled gas flow meters (Porter Instrument Co., Hatfield, PA) to obtain the desired NO ⅐ gas concentration.
Reactor-The reactor was an ultrafiltration cell modified as described previously and shown schematically in Fig. 1. For a detailed description, see Ref. 1. The only difference is the absence of the chemiluminescence detector to monitor NO ⅐ . Briefly, the reactor was a modified 200-ml stirred ultrafiltration cell (Amicon Model 8200) to which gas inlet and outlet ports, two ports for a flow loop connected to a spectrophotometer (for NO 2 Ϫ and NMor measurements) and a thermometer were added. A needle was inserted into the gas outlet port for argon/NO ⅐ delivery and removed during the reaction. The reactor was at ambient temperature.
N-Nitrosation of Morpholine-Morpholine at 50 -1500 M was added to 150 ml of buffer in the reactor, and the pH was measured. Stirring was initiated at 40 rpm and circulation through the flow loop was started. The solution was bubbled with argon for 45 min, and a mixture of NO ⅐ /Ar was then introduced for 30 min at 350 sccm to obtain the desired aqueous NO ⅐ concentration. Bubbling of the NO ⅐ /Ar mixture was terminated, and residual NO ⅐ in the head space was removed by introducing argon via the gas inlet for 1.5 min. A 21:79 mixture of O 2 and N 2 was then introduced through the gas inlet at the same flow rate (350 sccm). Diffusion of O 2 into the aqueous phase initiated the oxidation of NO ⅐ which at this time was approximately 25 M. The reaction was allowed to proceed for 30 min during which NO 2 Ϫ and NMor concentrations were monitored. Upon completion of the reaction, the pH was measured.
Nitrite and N-Nitrosomorpholine Analysis-The aqueous solution was continuously circulated at 45 ml/min through the 1/8-inch diameter flow loop (volume ϳ10 ml) and into a 10-mm spectrophotometer flow cell (Hewlett Packard, Model HP8452A UV) using a pulseless pump (Cole Parmer, Chicago, IL). The absorbance was measured at intervals of 1 min. Absorbances at 250 nm were linearly proportional to the NMor concentration (⑀ ϭ 5500 M Ϫ1 cm Ϫ1 ) with no interference from NO 2 Ϫ . The nitrite ion concentration was proportional to the absorbance at 210 nm (⑀ ϭ 5200 M Ϫ1 cm Ϫ1 ) although absorption of NMor at 210 nm necessitated a correction of Ϫ0.27 M NO 2 Ϫ /M NMor.

Concentrations of NMor and NO 2
Ϫ were calculated at each cycle point for the 30-min reaction.
Kinetic Model and Reaction Scheme-In previous experiments with this reactor, Lewis et al. (1) showed that the principal nitrosating agent in the NO ⅐ oxidation pathway at physiological pH is N 2 O 3 which leads primarily to NO 2 Ϫ as summarized in reactions 1-3.
Morpholine nitrosation by N 2 O 3 and enhanced hydrolysis of N 2 O 3 by various anions (X Ϫ ) are summarized in Equations 4 and 5.
Specifically, phosphate and chloride react with N 2 O 3 as shown below (1): Any anion that behaves in this manner will scavenge some of the N 2 O 3 , thereby decreasing the rate of N-nitrosation at neutral pH.
In the above reaction scheme, all reaction rate constants are known. The overall nitrogen balance performed by Lewis et al. (1) confirms that pseudo-steady state approximations for NO 2 , N 2 O 3 , and XNO are valid. A detailed analysis of the conservation equations at physiological pH is given in Ref. 1. The overall reaction kinetics where N 2 O 3 is the only significant nitrosating agent are summarized by the following equations: and, where the summation in Equation 9 is over all participating anions. For those anions that react with N 2 O 3 , the lumped "constant" k* will depend inversely on the anion concentration. By measuring k* in the presence of several concentrations of participating anions, the rate constants were determined for the reaction of phosphate and chloride with N 2 O 3 (1). In the presence of phosphate and one additional anion, rearrangement of Equation 9 yields: Further rearrangement gives: Using the data for phosphate alone, linear regression of 1/(2k*[P i ]) versus 1/[P i ] yields k 10 Pi /k 6 and k 4 /k 6 as the intercept and slope. The values of k 6 /k 4 for morpholine and k 10 Pi /k 4 were found in this way (1). In this work, k 6 /k 4 for morpholine and k 10 Pi /k 4 were determined as described above. In addition, the data for solutions containing bicarbonate and phosphate were used to calculate the rate constant for the reaction of bicarbonate with N 2 O 3 at pH 8.9 (k 10 HCO3 Ϫ /k 4 ).

RESULTS
Morpholine Concentration and pH-The unprotonated form of morpholine is the substrate for nitrosation and is thus the most important form of morpholine for these experiments. Denoting total morpholine as Mor and the unprotonated form as Mor 0 , the respective concentrations are related by: where the pK at 25°C is 8.5 for morpholine. The amount of morpholine available for nitrosation is 7.4% and 71.5% of the total morpholine concentration at pH 7.4 and 8.9, respectively. In phosphate buffer, the pH was nearly constant during a given experiment. In the bicarbonate reactions, the pH of the buffer rose during degassing to approximately 8.9. However, the pH during the reaction itself (i.e. after O 2 addition) remained virtually constant. The concentration of unprotonated morpholine therefore did not change significantly during the reaction. In all experiments, some NMor and NO 2 Ϫ were present in the solution prior to introduction of O 2 due to a small air leak in the flow loop that could not be eliminated.
Effect of Hydroxide Ion Concentration on Nitrosation Kinetics-It has been reported that OH Ϫ enhances the rate of hydrolysis of N 2 O 3 (13). Using flash photolysis of NO 2 Ϫ ions in the presence of NO ⅐ in the pH range 9 -10, a factor for total N 2 O 3 hydrolysis was reported to be 2000 s Ϫ1 ϩ 10 8 [OH Ϫ ] M Ϫ1 s Ϫ1 representing terms for both water and hydroxide-induced hydrolysis of N 2 O 3 (13). In order to determine the rate constant for reaction of hydroxide with N 2 O 3 under the present conditions, morpholine nitrosation reactions were performed at several pH values in the range pH 7.4 -8. There is some ambiguity in the meaning of k 4 depending on whether the hydroxide contribution is included. However, the hydroxide term does not significantly affect the k 4 value at pH 7.4 due to the extremely small concentration of hydroxide at this pH. For example, if a value for k 4 is assumed to be 1600 s Ϫ1 at pH 7.4 (1), the incremental increase for the hydroxide contribution is 150 s Ϫ1 . Given the wide range of reported k 4 values as discussed by Lewis et al. (1), this 10% difference does not seem to be very important. The rate constants here are expressed as ratios to k 4 .
Effect of Phosphate on N-Nitrosation at pH 8.9 -To assess the validity of these experiments at pH 8.9, the rate constants for reaction of morpholine and phosphate with N 2 O 3 relative to N 2 O 3 hydrolysis (k 6 /k 4 and k 10 Pi /k 4 ) were measured and compared to the published results. Various morpholine concentrations (50 M to 150 M) were used in buffers of three different phosphate concentrations, 0.01 M, 0.025 M, and 0.05 M.
As seen in Fig. 2, the marked decrease in slope of ⌬[NMor]/ ⌬[NO 2 Ϫ ] Ϫ ⌬[NMor] versus [Mor 0 ] with increasing phosphate concentration indicates that phosphate inhibits nitrosamine formation. When the slope was calculated by linear regression using the average data between 3 and 30 min, values of k* at pH 8.9 for 0.01 M, 0.025 M, and 0.05 M phosphate were 1500 Ϯ 300 M Ϫ1 , 1100 Ϯ 100 M Ϫ1 , and 600 Ϯ 100 M Ϫ1 , respectively. The differences between these values and those previously published are due to the effect of hydroxide at pH 8.9. The rate constants for the reaction of N 2 O 3 with phosphate and morpholine at pH 8.9 relative to N 2 O 3 hydrolysis (k 10 Pi /k 4 and k 6 /k 4 ) were calculated from a plot of the equation below: The intercept (k 10 Pi /k 6 ) was found to be (1.1 Ϯ 0.2) ϫ 10 Ϫ2 which agrees nicely with the previously reported value of 1.0 ϫ 10 Ϫ2 (1). The value of the rate constant for the phosphate/N 2 O 3 reaction relative to N 2 O 3 hydrolysis (k 10 Pi /k 4 ) was then calculated to be 4.0 ϫ 10 2 M Ϫ1 . The rate constant for the morpholine/ N 2 O 3 reaction relative to N 2 O 3 hydrolysis (k 6 /k 4 ) was determined from the slope to be addition of 0.04 M sodium bicarbonate. Using linear regression, the value of k* using the average data between 3 and 30 min for reactions containing 0.01 M phosphate and 0.04 M bicarbonate at 25°C and pH 8.9 was 400 Ϯ 60 M Ϫ1 . The rate constant for the bicarbonate/N 2 O 3 reaction relative to N 2 O 3 hydrolysis (k 10 HCO3 Ϫ /k 4 ) was calculated from Equation 10. The resulting k 10 HCO3 Ϫ /k 4 value is (9.3 Ϯ 1.5) ϫ 10 2 M Ϫ1 . The rate constants from this study are summarized in Table I, and their importance is discussed below.

DISCUSSION
The finding that N-nitrosation of morpholine is inhibited by bicarbonate provides an additional pathway that affects the fate of N 2 O 3 in vitro and in vivo. The rate constant for the bicarbonate/N 2 O 3 reaction relative to N 2 O 3 hydrolysis (k 10 HCO3 Ϫ /k 4 ) in oxygenated nitric oxide solutions is (9.3 Ϯ 1.5) ϫ 10 2 M Ϫ1 which is greater than the rate constant for the phosphate/N 2 O 3 reaction relative to N 2 O 3 hydrolysis (k 10 Pi /k 4 ) found to be (4.0 Ϯ 0.9) ϫ 10 2 M Ϫ1 . Inhibition by bicarbonate will be significant due to the higher rate constant for bicarbonate and higher extracellular concentrations of bicarbonate relative to phosphate. As summarized in Table I, the agreement between the published rate constants for the morpholine and phosphate reactions at pH 7.4 and the experimentally determined rate constants at pH 8.9 demonstrates the validity of performing these experiments at pH 8.9 and assures the applicability of these rate constants at pH 7.4. Further work is necessary to determine an exact rate constant for the bicarbonate/N 2 O 3 reaction at pH 7.4 to compare with that found here at pH 8.9. Nevertheless, it is certain that bicarbonate can efficiently scavenge the nitrosating agent N 2 O 3 in competition with nitrosation of morpholine.
The importance of including this previously unrecognized reaction of bicarbonate in nitrosation experiments can be demonstrated by analyzing recent work by Lewis et al. (14). In an effort to determine whether the rate constants of pertinent reactions measured in simple cell-free systems could account for the rates at which the products are formed in the presence of NO ⅐ -generating cells in complex media, they studied the formation of NMor by activated macrophages. The importance of buffer anions is emphasized by the fact that the predicted values for NMor formation were 28 times larger than measured levels of NMor without the inclusion of chloride and phosphate contributions to N 2 O 3 hydrolysis. The difference between observed and predicted values decreased to only 7-fold when these terms were included. This suggests that while chloride and phosphate are important, the cell culture system contains additional unrecognized compounds that can compete for N 2 O 3 . Bicarbonate is a prime candidate to account for at least a portion of the N 2 O 3 scavenged. In this case, N 2 O 3 hydrolysis by bicarbonate will be more significant than phosphate due to the much higher concentrations of bicarbonate (30 mM NaHCO 3 versus 0.9 mM P i ). Calculations show that the inclusion of a term for bicarbonate reduces the difference between observed and predicted values for NMor by an additional factor of 3.
The contribution to N 2 O 3 hydrolysis by the different anions can be roughly calculated given the rate constants and the media concentrations. In Lewis' kinetic analysis, a lumped constant k* is calculated from the measured concentrations of NMor and NO 2 Ϫ using Equation 8. This k* value is related to the rate constants for morpholine nitrosation (k 6 ) and several terms for N 2 O 3 hydrolysis (k 4 and k 10 X Ϫ ) shown below which can also be expressed relative to N 2 O 3 hydrolysis (k 4 ) as follows: The anion contribution to N 2 O 3 hydrolysis is therefore the summation over all anions, X Ϫ , which can be expanded to include terms for phosphate, chloride, bicarbonate, and remaining unidentified anions as follows: The contribution for the anions currently known to scavenge N 2 O 3 using the media concentrations for these experiments is shown here as a hydrolysis enhancement ratio representing the additional hydrolysis with the anion present: phosphate, (4.0 ϫ 10 2 M Ϫ1 ) (0.9 mM) ϭ 0.4; chloride, (9.0 ϫ 10 1 M Ϫ1 ) (110 mM) ϭ 9.9; bicarbonate, (9.3 ϫ 10 2 M Ϫ1 ) (30 mM) ϭ 27.9. Bicarbonate is extremely significant in this case and in all biological situations because it is present at relatively high concentrations. Physiological concentrations of phosphate, chloride, and bicarbonate in interstitial fluid are approximately 5 mM, 110 mM, and 30 mM, respectively, and the concentrations of these species in intracellular fluid are approximately 80 mM, 5 mM, and 12 mM, respectively (15). Predictions for contributions of these participating anions to N 2 O 3 hydrolysis in vivo were calculated using these concentrations for both the intracellular and extracellular environments and are shown below as hydrolysis enhancement ratios. The intracellular ratios are: phosphate, Bicarbonate's significant contribution to N 2 O 3 hydrolysis in the extracellular milieu demonstrated by the large hydrolysis enhancement ratio may be important in protecting cells from N 2 O 3 formed near NO ⅐ -producing cells such as macrophages during an inflammatory response. In this situation, N 2 O 3 would be scavenged in the extracellular fluid before encountering neighboring cells.
Therefore, bicarbonate which is present at high concentration in vivo will be a key determinant of the fate of NO ⅐ -derived reactive species due to its reaction with N 2 O 3 reported here and Ϫ the previously reported reaction with ONOO Ϫ . Carbonate has long been known to alter the activity of several types of oxygen radicals including superoxide anion, hydroxyl radical, and singlet oxygen (4). In addition, peroxynitrite has been known to be unstable in carbonate buffers for many years (12). Recently, it has been demonstrated that bicarbonate does indeed affect peroxynitrite reactivity (2,3). Bicarbonate inhibits the toxicity of peroxynitrite to Escherichia coli (2) which may be a direct result of the enhanced isomerization to nitrate leading to ONOO Ϫ decomposition before it can encounter the bacteria. Extremely low levels of bicarbonate (far below physiological concentrations) are required as demonstrated by the fact that 95% protection from toxicity is observed at 5 mM bicarbonate (2). The probable mechanism involves reaction of carbon dioxide with peroxynitrite forming the nitrosoperoxycarbonate anion, OϭN-OOCO 2 Ϫ (3, 5, 6). Carbon dioxide increases the rate of peroxynitrite isomerization to nitrate presumably through this species (5). The result is ONOO Ϫ scavenging which will be important in defining amounts of tissue injury including both oxidation and nitration products resulting from peroxynitrite (16 -18).
Elucidation of the exact reaction pathways and rate constants for the reaction of bicarbonate/CO 2 with peroxynitrite will provide additional information about the fate of NO ⅐ in specific systems. Then, reactions of NO ⅐ with oxygen and superoxide can be modeled in detail. The newly determined rate constant for the bicarbonate/N 2 O 3 reaction will contribute to kinetic modeling ultimately enhancing the understanding of the numerous biological roles of NO ⅐ both as a messenger and as a cytotoxic or mutagenic agent.