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Chem. Res. Toxicol. 1997, 10, 408-413
Measurement and Modeling of Nitric Oxide Release Rates for Nitric Oxide Donors Anand Ramamurthi and Randy S. Lewis* School of Chemical Engineering, Oklahoma State University, Stillwater, Oklahoma 74078 Received November 5, 1996X
An accurate model of the nitric oxide (NO)-release rate is essential for predicting the temporal NO-release rate and resulting NO concentrations for NO donors. Knowledge of the NO-release rate and/or the NO concentration is beneficial for assessing the physiological or pathological effects of NO on cell systems. This study describes a method to measure the temporal NOrelease rate from NO donor compounds utilizing a modified ultrafiltration cell. For this study, the NO-release rates of spermine NONOate and diethylamine NONOate were measured and kinetically modeled at pH 7.4 and 37 °C. An advantage of this method is that complete dissolution of the NONOate was not necessary for modeling the NO-release rate. One model parameter, which is the number of moles of NO released per mole of decomposed NONOate, is 1.7 ( 0.1 and 1.5 ( 0.2 for spermine and diethylamine NONOate, respectively. The other model parameter, which is the NONOate first-order decomposition rate constant, is 0.019 ( 0.002 min-1 and 0.47 ( 0.10 min-1 for spermine and diethylamine NONOate, respectively, as determined from NO concentration profiles. The decomposition rate constant measured by spectrophotometry was consistent with the above value for spermine NONOate but was approximately half the above value for diethylamine NONOate. Preliminary experiments using spectrophotometry showed that for both NONOates the decomposition activation energy was ∼100 kJ/mol. The NO-release rate model for spermine NONOate was applied to a model for predicting the NO concentration in oxygenated solution. The NO concentration was measured in phosphate buffer, culture medium, and Tyrode’s solution. Excellent agreement was observed between experimental and predicted NO concentrations.
Introduction Nitric oxide (NO),1 which is synthesized endogenously by various cells including macrophages, neutrophils, endothelial cells, and hepatocytes, is a biological molecule involved in several physiological and pathological processes (1). Physiological roles include blood pressure regulation, neurotransmission, and inhibition of platelet adhesion. It is also involved in immunological responses as an important cytotoxic molecule and precursor. Excessive production of NO has been implicated at least partly in hypotension, inflammation-associated tissue damage, rheumatoid arthritis, and insulin-dependent diabetes mellitus. On the other hand, diminished NO production has been implicated in pulmonary hypertension, arteriosclerosis, and reperfusion injury (for review, see refs 2-5). Compounds which release NO have a potential benefit for releasing NO to NO-deficient areas. Several NOreleasing compounds are available, including NONOates (6), SIN-1 (7), and molsidomine (7). For applications which utilize NO-releasing compounds, it is critical to control the NO-release rate since excessive exposure to NO or its reactive products can be cytotoxic, carcinogenic, or mutagenic (8-10). Therefore, a proper model of the NO-release rate is essential for predicting the temporal NO-release rate and NO spatial concentration when seeking to minimize the undesired effects of NO or when assessing the effects of NO on cell systems. * Address correspondence to this author at the School of Chemical Engineering, 423 EN, Oklahoma State University, Stillwater, Oklahoma 74078. Telephone: (405) 744-5280. FAX: (405) 744-6338. Email:
[email protected]. X Abstract published in Advance ACS Abstracts, March 15, 1997. 1 Abbreviation: NO, nitric oxide.
S0893-228x(96)00183-X CCC: $14.00
A method is described in which the temporal NOrelease rate is obtained from NO concentration data. Real-time NO concentrations are measured utilizing a modified ultrafiltration cell. Kinetically or empirically modeling the NO-release rate is then feasible using the NO-release rate data. An advantage of this method is that complete dissolution of the NO-donor compound is unnecessary for measuring and modeling the NO-release rate. For this study, spermine NONOate and diethylamine NONOate were utilized for demonstrating the method of measuring and modeling the temporal NOrelease rate. A previous method for modeling the NO-release rate of NONOates required complete decomposition of the NONOate (6). For such conditions, the NO-release rate of spermine NONOate was not characterized at physiological pH since the NONOate decomposition was too slow. For this study, NO-release rate model parameters using a previously developed kinetic mechanism were obtained for spermine NONOate and diethylamine NONOate at physiological pH. For spermine NONOate, model parameters were obtained without complete NONOate decomposition. Modeling of the NO-release rate is beneficial for predicting the temporal NO-release rate and NO concentration in a properly characterized system. The NOrelease rate model of spermine NONOate was used to predict the NO concentration in several oxygenated solutions. Excellent agreement between the predicted and measured NO concentration showed the validity of applying an NO-release rate model to a well-defined experimental system. Thus, the process of measuring and modeling the NO-release rate, as well as applying the NO-release rate to an experimental system, is demonstrated in this study. © 1997 American Chemical Society
Characterizing Nitric Oxide Release Rates
Materials and Methods Caution: All experiments were performed in a certified hood due to the potential toxicity of NO. Reagents. Ultra-high-purity nitrogen, following passage through an oxygen trap, was mixed with pure NO using controlled gas flow meters (Porter Instrument Co., Hatfield, MA) to obtain the desired NO gas concentration. Following mixing, the NO/N2 mixture was passed through a column of soda lime and bubbled through 0.1 M sodium hydroxide to remove NOx impurities. Air or 5% CO2-balance air were the sources of oxygen. Phosphate buffer (0.1 M, pH 7.4) was prepared from sodium diphosphate and sodium monophosphate. Culture medium (pH 7.4) was composed of D-MEM, 3.7 g of sodium bicarbonate, 1 mM sodium pyruvate, 20 mM HEPES buffer, and 10% donor calf serum. Tyrode’s albumin buffer (pH 7.35) contained 154 mM NaCl, 2.7 mM KCl, 12 mM NaHCO3, 0.35 mM Na2HPO4, 1.2 mM MgCl2, 2 mM CaCl2, 5.5 mM glucose, 0.35% bovine albumin, and 2.5 mM HEPES. D-MEM (without phenol red, sodium pyruvate, or sodium bicarbonate), donor calf serum, and HEPES were obtained from Life Technologies (Grand Island, NY). Glucose, sodium pyruvate, and bovine albumin (Ca2+ and fatty acid free) were obtained from Sigma (St. Louis, MO). Spermine NONOate and diethylamine NONOate were obtained from Cayman Chemical (Ann Arbor, MI). NONOate Preparation. For deoxygenated experiments, NONOate samples were dissolved in alkaline buffer (pH 12.0) prepared from 0.1 M sodium phosphate (dibasic) and 0.1 M NaOH prior to injection. NONOate samples were dissolved in 0.01 M (pH 12.0) buffer for oxygenated experiments. No differences in the NONOate dissolution kinetics were observed between the two alkaline buffer ionic strengths. In all cases, 10 mg of NONOate was dissolved in an appropriate volume of the pH 12.0 buffer to obtain desired NONOate concentrations for injection. Experimental Apparatus. A modified 200 mL ultrafiltration cell (Amicon Inc., Beverly, MA, Model 8200) was used to detect the aqueous NO concentration as previously described (11). Briefly, a semipermeable membrane (MEM-100, Membrane Products, Albany, NY) laminated to a Teflon sheet containing 24 symmetrically spaced holes (6.8 cm2 total area of holes) was placed at the base of the ultrafiltration cell. Solution was placed in the ultrafiltration cell, and vacuum from a chemiluminescence detector (Seivers Instruments, Boulder, CO, Model NOA 270B) was applied to the bottom side of the membrane. The holes in the Teflon permitted the transport of NO from the solution and into the detector. A septum port for NONOate injection, inlet, and outlet ports for purging of the gas head space, and a thermometer were also included. For experiments with phosphate buffer, stirring was maintained at 930 rpm with a magnetic stir bar supported by a Teflon cage. In addition, a flow loop (1/8 in. diameter, 12 mL total volume) containing a gear pump (Micropump, Vancouver, WA, Model 000-305) was incorporated to measure the disappearance of the NONOate compound using a UV-spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD; Model UV 1601-PC). The flow rate through the loop was maintained at 70 mL /min. For experiments with culture medium or Tyrode’s buffer solution, stirring was maintained at 85 rpm using a stirrer from a Cytostir stirred bioreactor (Kontes, Vineland, NJ) to minimize protein damage. The flow loop was not incorporated for these latter experiments. All studies were at 37 °C. Nitric Oxide Calibration. Phosphate buffer (80 mL), added to the modified ultrafiltration cell, was bubbled with a NO/N2 gas mixture following deoxygenation. Bubbling was achieved by flowing gas through a needle inserted into the solution via the septum port. The saturated NO concentration was calculated from solubility data (12). The detector responses for NO concentrations up to 10 µM were measured. The response was linear with concentration with a minimum detection limit of approximately 30 nM. Mass Transfer Coefficient Determination. Since NO leaves solution during an experiment, it is important to quantify the rate at which NO transports from solution. The NO
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 409 volumetric mass transfer coefficient (kLa/V), which characterizes this transport rate into both the head space and detector, was measured in phosphate buffer (92 mL total volume) using a previously described method (11). The value of kLa/V was 0.29 ( 0.01 min-1 at 930 rpm with the flow loop included and 0.10 ( 0.001 min-1 at 85 rpm without inclusion of the flow loop. NO-Release Rate Characterization. For measuring and modeling the NO-release rate of spermine NONOate or diethylamine NONOate, 92 mL of phosphate buffer was added to the modified ultrafiltration cell and flow loop. The solution was deoxygenated by purging the head space with N2 for 1 h. Following purging, spermine NONOate (0.5 mL) or diethylamine NONOate (0.1 or 0.2 mL) dissolved in alkaline solution was injected into the phosphate buffer to initiate NONOate decomposition and NO generation. Spermine and diethylamine NONOate alkaline solutions contained 0.33 and 0.1 mg/mL, respectively. The NONOate concentration was spectrophotometrically measured at 60 or 30 s intervals for spermine or diethylamine NONOate, respectively. NONOate concentrations were determined at 250 nm using extinction coefficients of 8000 and 6500 for spermine and diethylamine NONOates, respectively, as reported by Maragos et al. (6). The aqueous NO concentration was continuously measured in all experiments. Nitric Oxide Concentration Predictions in Oxygenated Solutions. The validity of a model to predict the aqueous NO concentration following the addition of spermine NONOate to oxygenated solution was assessed by continuously measuring the aqueous NO concentration. Oxygenated solution was used to induce reaction with NO. In addition, it is also more relevant to physiological conditions. Phosphate buffer, culture medium, or Tyrode’s buffer solution was added (92 mL) to the modified ultrafiltration cell, and in the case of phosphate buffer, to the flow loop in addition. Air for phosphate buffer studies or 5% CO2-balance air for culture medium or Tyrode’s solution studies was purged through the head space for 1 h prior to the spermine NONOate injection. Purging was then maintained throughout the experiment. For phosphate buffer studies, injection volumes of spermine NONOate alkaline solution were 0.2 mL containing 10 mg/mL, 0.5 mL containing 0.33 mg/mL, or 0.25 mL containing 0.33 mg/ mL. Therefore, following injection the initial spermine NONOate concentrations were predicted as 82.7, 6.9, or 3.45 µM, respectively. For culture medium or Tyrode’s solution, the injection volume was 0.2 mL containing 10 mg/mL, corresponding to a predicted initial spermine NONOate concentration of 82.7 µM. Prior to and following spermine NONOate introduction, the aqueous NO concentration was continuously measured. Following each experiment, the pH was measured.
Results and Discussion NO donor compounds can be assessed for their temporal NO-release rates by measuring temporal NO concentration profiles in solution. Modeling the NOrelease rate is beneficial for predicting NO concentrations or NO-release rates for various experimental systems. The results of measuring and modeling the NO-release rates for spermine NONOate and diethylamine NONOate are described below. In addition, the validity of utilizing the NO-release rate model for spermine NONOate in predicting NO concentrations in a well-defined oxygenated system is also assessed. Nitric Oxide Profiles. The aqueous NO concentration was continuously measured in 0.1 M deoxygenated phosphate buffer solution following the injection of spermine NONOate or diethylamine NONOate. Table 1 gives the maximum NO concentrations observed and the initial NONOate concentrations for each experiment. The initial NONOate concentrations were based on spectrophotometric measurements using molar extinction coefficients of 8000 and 6500 for spermine and diethylamine
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Ramamurthi and Lewis
Nitric Oxide Release Rates. The NO-release rate as a function of time can be determined from NO versus time data when applied to the conservation of mass equation for NO. For a well-stirred solution with a NOfree gas head space, the conservation equation is
d[NO] ) NO release rate - 4k*[NO]2[O2] dt (kLa/V)[NO] (1)
Figure 1. Dimensionless NO profiles for spermine (n ) 8) and diethylamine (n ) 5) NONOates. Means ( SD are shown as discrete symbols for all deoxygenated experiments in 0.1 M phosphate buffer at pH 7.4 and 37 °C. Solid lines represent the dimensionless NO profiles predicted by eqs 1 and 6 with k1 ) 0.019 min-1 for spermine NONOate and k1) 0.47 min-1 for diethylamine NONOate. The dashed line represents the prediction with k1) 0.25 min-1 for diethylamine NONOate. Table 1. Initial NONOate and Peak Nitric Oxide Concentrations spermine
diethylamine
[NONOate]0 (µM)
[NO]max (µM)
[NONOate]0 (µM)
[NO]max (µM)
6.13 6.22 6.25 6.53 5.95 6.25 6.41 5.70
0.52 0.52 0.55 0.72 0.51 0.56 0.52 0.51
0.66 1.24 1.30 1.46 1.31
0.48 0.96 1.04 0.81 0.85
NONOates, respectively (6). For spermine NONOate, initial NONOate concentrations based on calculations using injected amounts agreed closely (
