In the Classroom
Relevance of Chemical Kinetics for Medicine: The Case of Nitric Oxide Alexandru T. Balaban* and William Seitz Department of Marine Sciences, Texas A&M University at Galveston, Galveston, TX 77553; *
[email protected] It is well known that nitric oxide, NO, is a reactive free radical that reacts readily with elemental oxygen, which is a diradical. The stoichiometric equation of this autoxidation is
2 NO + O2
2 NO2
(1)
When the reactants and the products are in gas phase at 25 ⬚C, the rate of this exothermic reaction is
−d [NO] 2 = k [NO] [O2 ] dt
(2)
with specific rate constant of k = 1.42 × 104 M᎑2 s᎑1 (1). In aqueous solution at the same temperature, the specific rate constant is 8 × 106 M᎑2 s᎑1 (2). Thermodynamically, at 25 ⬚C the equilibrium for eq 1 is completely shifted to the right, but at higher temperatures (over 600 ⬚C) the reverse reaction becomes favored for entropic reasons. The enthalpy and free energy of formation for NO are 90.2 and 86.6 kJ兾mol, respectively, indicating that NO is thermodynamically unstable, like all other nitrogen oxides. In many situations, oxygen is present in excess so that its concentration does not change appreciably as the result of the autoxidation. In this case eq 2 becomes a simple bimolecular (or more appropriately termed pseudobimolecular) reaction. The usual integration of eq 2 with k´= k[O2] yields
1
[NO]
−
1
[NO]0
= k' t
(3)
where [NO]0 is initial concentration of nitric oxide and t is the reaction time. The half-life is then given by setting [NO] = 1/2[NO]0 to give t1 2 =
1 k' [NO]0
(4)
Later we will illustrate the importance of the inverse relation between half-life and concentration in determining the utility of nitric oxide in biological systems, but first we review some background on nitric oxide both as an industrial pollutant and as a useful (and now almost ubiquitous) biomolecule. The production of nitric acid (about 20 billion pounds per year in the United States alone) requires enormous quantities of nitric oxide, which is manufactured on a large scale via the Wilhelm Ostwald process by oxidizing ammonia with oxygen. With an excess of oxygen, as one sees when a stream of gaseous NO is allowed to escape in the atmosphere, this highly concentrated nitric oxide reacts according to eq 1 practically instantaneously yielding nitrogen dioxide, NO2, a redorange, very toxic gas with a penetrating characteristic smell. Sometimes one sees reddish plumes of nitrogen dioxide ema662
nating from stacks of large production plants for nitric acid to be used in the production of nitrogen fertilizers in Eastern Europe and Russia. When high concentrations of NO and NO2 are present, these free radicals in turn combine reversibly with a high reaction rate forming dinitrogen trioxide (nitrous acid anhydride),
NO + NO2
N2O3
(5)
The enthalpy and free energy for eq 5 are ᎑40.1 and 1.84 kJ兾mol, respectively, so that at 25 ⬚C about 10% N2O3 is formed in the gaseous equilibrium mixture starting from equimolar amounts of reactants. Lowering the temperature increases the amount of N2O3. During the last ten years, nitric oxide has been shown to possess a remarkable and at first surprising role in the biology of mammals, including humans—surprising in that it is known to generate harmful NO2. Nevertheless, NO is continuously synthesized from arginine by the enzyme nitric oxide synthase, e-NOS, in the endothelial cells lining blood vessels (arteries, veins, etc.). It is now believed that NO in strictly regulated concentrations is responsible for maintaining proper vasodilation (blood pressure). Any NO deficiency, which increases with advancing age, causes hypertension. Nitric oxide has also been found to act as a neuronal mediator in the brain, where it is produced by another enzyme, neuronal n-NOS. These two constitutive NOS isoforms maintain low concentrations of NO. Larger amounts of NO are released by a third enzyme, the inducible isoform i-NOS, which is present in macrophages and plays a beneficial role in killing invading microorganisms, but may become lethal when it is overproduced in shock. Several books on NO have recently been published (3– 8) and in 1992 Science advertised NO as the molecule of the year. The Nobel Prize for Medicine or Physiology was awarded in 1998 to R. L. Furchgott of the State University of New York in Brooklyn (9), L. J. Ignarro of the University of California at Los Angeles (10), and F. Murad of The University of Texas Medical School in Houston (11) for their discoveries concerning nitric oxide, which was first known as endothelium-derived relaxing factor (12–15). It is now believed that NO is carried in living cells via S-nitroso groups attached to thiols such as glutathione and via coordination compounds between NO and iron atoms in iron-porphyrins such as the heme of hemoglobin. Although the beneficial effects of glycerol trinitrate have been known for more than a century (Alfred Nobel’s angina could have been treated with this compound had he allowed it, the same unpredictable explosive that he had “tamed” by inventing dynamite), only recently has it become clear that such nitrate esters act by donating NO. Research on the biological activity and applications of NO has led to the publication of thousands of papers per year. A specialized journal,
Journal of Chemical Education • Vol. 80 No. 6 June 2003 • JChemEd.chem.wisc.edu
In the Classroom
Nitric Oxide, began publication a few years ago. The present authors and their colleagues were involved in the synthesis of new NO donors related to cupferron (16), the possibility of having solutions of NO in perfluorocarbon emulsions (17), and the topical delivery of NO by admixing two gels (18). Returning now to kinetics, at high concentrations, NO reacts rapidly with oxygen, but the reaction is very slow at very low concentrations. This effect is quantified by eqs 2, 3, and 4. At normal oxygen concentrations (as in air or in saturated aqueous solutions), but at low NO concentrations (such as generally found biologically), the reaction becomes pseudo-bimolecular in NO and eq 4 can be used. Recall that the volume of one mole of an ideal gas at 25 ⬚C and at one atmosphere is approximately 24 L. The concentration of oxygen in air is about 1兾5 = 20%, that is, 200,000 parts per million or ppm. In terms of mol兾L (i. e., M), this corresponds to 1兾(5 × 24) = 8.3 × 10᎑3 M. When the concentration of NO is 1% or lower, this oxygen concentration remains essentially constant. For example, suppose that a mixture of air and a small amount of nitric oxide (say, 1% = 10,000 ppm) is allowed to react at 25 ⬚C. Then, [O2] = 0.20兾24 = 8.3 × 10᎑3 M (hence k´ = 120 M᎑1 s᎑1) and [NO]0 = 0.010兾24 = 4.2 × 10᎑4 M, and t / , obtained from eq 4, is found to be about 20 s. Now, lower the NO concentration 1,000 times, reaching 10 ppm. Since the half-life t / is inversely proportional to the concentration of NO, it increases to 20,000 seconds or almost 6 h. As a second example, consider an aqueous solution saturated with oxygen, [O2] = 4.6 × 10᎑4 M, and with a concentration of NO = 100 mM at 25 ⬚C. Again, eq 4 can be utilized (with the proper aqueous rate constant) to give a half-life t / = 4.5 s. But at 1 mM concentration, t / = 450 s or about 7.5 min, and at 10 nM concentration, which is sufficient for 50% blood vessel relaxation, t / ≈ 13 h. Actually, in the lungs NO traverses the alveoli rapidly and, after relaxing the blood vessels, coordinates irreversibly with the divalent iron in iron-porphyrins, especially with oxyhemoglobin (with a rate constant that is 20 times larger than the rate of autoxidation; ref 19). Thus, only the lung blood vessels feel the relaxing effect of nitric oxide. The removal of NO via this process before significant formation of NO2 occurs is extremely important biologically. NO2 is a powerful oxidant that converts Fe(II) into Fe(III) affording methemoglobin, and in addition reacts with water yielding nitrous and nitric acids that cause pulmonary edema. In fact, breathing NO2 at concentrations as low as 100 ppm for a few minutes can be lethal. Burning fuel or smoking cigarettes causes the formation on nitrogen oxides, NOx, as well, and the U.S. Occupational Safety and Health Administration has set the average nitric oxide value for 8 h at a maximum level of 25 ppm (20). Smokers inhale NO at concentrations of 400 to 1000 ppm, and the vasodilatation effect may facilitate the penetration of carcinogens as well as potentially forming toxic NO2. The first studies for using NO to facilitate gas transfer in lungs by dilating the blood vessels were performed about ten years ago with newborn lamb (21–23). Then clinical studies followed rapidly, mainly for newborns and adults with acute respiratory distress syndrome, hypoxic respiratory failure, and persistent pulmonary hypertension (24–30). For its bronchodilatory effect, NO is administered to newborn in1
2
1
1
1
2
1
2
2
fants or patients with pulmonary problems, admixed with air or oxygen-enriched air, at NO concentrations of less than 80 ppm. The ventilator used in hospitals carries a freshly prepared mixture of air with about 10–80 ppm of NO to the nose and mouth of the patient (31–34). The mean transit time from the gas reservoir bag to the patient’s pulmonar alveoli is less than 30 s. In these conditions, less than 4 ppm of NO2 will be inhaled. However, it is mandatory to monitor precisely the concentrations of NO and NO2 with fluorescence detectors, so that these concentrations are always kept at the corresponding levels. Conclusion The autoxidation of NO to yield the toxic NO2 is a second-order reaction in NO that is so slow at very low NO concentrations that NO is an important natural biomolecule. It is possible to find safe and beneficial uses for NO as a drug, for example, for relaxing blood vessels in patients with pulmonary problems. The progress from experiments with lambs to clinical trials and finally to current treatments for human ailments has been remarkably rapid. Further work is ongoing at universities and in pharmaceutical firms on many other NO systems for use in medicine. In all of this work, the kinetics of the formation of NO2 must be considered to ensure safe and effective nitric oxide therapies. For other applications of nitric oxide, see an excellent review published in this Journal (34) or an article concerning NO involvement in wound repair (35). Acknowledgment The financial assistance of the Welch Foundation of Houston is gratefully acknowledged.
2
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