(T1430-09-51) Feasibility of Controlled Nitric Oxide Generation via Ascorbate Induced Chemical Reduction of Nitrite Ions

Purpose: Due to its vasodilatory and antimicrobial effects, nitric oxide (NO) is a vital endogenous molecule and a potent therapeutic agent. Inhaled NO (iNO) is a lifesaving and FDA-approved drug to improve oxygenation in persistent pulmonary hypertension of the newborn (PPHN) as well as a potent antimicrobial agent. Its wider therapeutic application is, however, hindered due to the high-cost and logistical barriers of traditional NO/N2 gas tanks, emphasizing the importance of developing low-cost and portable tankless NO generators. Here, we explored the use of ascorbate to reduce nitrite ions via a copper-ligand mediator in the bulk solution phase for generating controlled levels of NO on-demand. We conducted a series of experiments to develop and characterize this approach.

Methods: The constituents of the receiver electrolyte solution used to generate NO were 7 mM 1,4,7-trimethyl-1,4,7-triazacyclononane (L), 7 mM copper sulfate (CuSO₄), and 140 mM calcium ascorbate in pH 5.8 MES solution. 10 mM sodium nitrite (NaNO₂) was injected into the receiver solution with a programmable syringe pump. NO was generated into 210 mL/min N₂ flow and detected with an ozone chemiluminescence NO analyzer (Sievers).

Results: The main parameters considered for optimization were the kinetics of NO formation, conversion efficiency of NO₂^– to NO and the shelf life of the electrolyte. The controlled infusion of NO₂^– ions into electrolyte containing 7 mM CuL and 140 mM calcium ascorbate rapidly generated NO and modulating the infusion of NO₂^– allowed for excellent controllability of the generated NO levels. Calcium was used to remove oxalate, a decomposition product of ascorbate, which was shown to inhibit the production of NO, from the solution. This improved the storage stability of the electrolyte; however, we observed the lowest nitrite to NO conversion with freshly made electrolyte (69% for 140 mM calcium ascorbate composition), a behavior that seemed contradictory to to our earlier finding that a degradation product of ascorbate interferes with NO generation. We investigated this apparent contradiction further by identifying the solid formed in aged electrolyte as calcium oxalate with ATR-FTIR and by titrating the electrolyte with calcium ions and with oxalate ions. At high concentrations of calcium ( >1 M), NO generation was suppressed. At oxalate concentrations greater than equimolar with CuL, NO generation rapidly dropped off (63% decrease in NO generation), although an initial, slight increase (21% increase) in NO generation was observed at low concentrations ( < 7 mM) of oxalate. Ascorbate decomposition is complex and highly situationally dependent. We proposed a decomposition pathway for our conditions that can explain the low nitrite conversion observed with fresh electrolyte as well as the initial slight increase in nitrite conversion (Figure 1). If the hydrolytic decomposition reactions were to exist in equilibria with each other, then a small amount of oxalate could actually produce conditions that favor an additional redox reaction in the decomposition of 2,3-diketo-L-gulonate (DKG) into 4-O-oxalyl-threonate. This additional reaction could explain the slight increase in NO generation observed with small amounts of oxalate, and removal of oxalate could result in bypassing that redox reaction. To provide further evidence for our proposed pathway, we investigated the nitrite conversion of equimolar ascorbate and CuL systems in the presence and absence of calcium ions and spiked the solutions with oxalate and threonate. Threonate is another later decomposition product of ascorbate. We found that excess calcium ions produced the lowest nitrite to NO conversion (21%), and threonate produced the highest 56%). While these results do support our proposed decomposition pathway, there is still an unaccounted mass balance deficit of nitrite. We analyzed the gases produced with our electrolyte system using FTIR and found that, under the investigated conditions (7 mM CuL, 3.5 mM calcium ascorbate and 14 mM CaCl₂), some of the gas produced was N₂O.

Conclusion: These data demonstrate its feasibility as an alternate cost-effective, and portable NO generation method by controlled mixing of two reactive solutions. Further work is needed to implement this method of ascorbate assisted copper-ligand mediated NO generation for clinically relevant flow rates.

References: (1) Qin, Y.; Zajda, J.; Brisbois, E. J.; Ren, H.; Toomasian, J. M.; Major, T. C.; Rojas-Pena, A.; Carr, B.; Johnson, T.; Haft, J. W.; Bartlett, R. H.; Hunt, A. P.; Lehnert, N.; Meyerhoff, M. E. Portable Nitric Oxide (NO) Generator Based on Electrochemical Reduction of Nitrite for Potential Applications in Inhaled NO Therapy and Cardiopulmonary Bypass Surgery. Mol. Pharm. 2017, 14 (11), 3762–3771. https://doi.org/10.1021/acs.molpharmaceut.7b00514.
(2) Oh, B. K.; Meyerhoff, M. E. Catalytic Generation of Nitric Oxide from Nitrite at the Interface of Polymeric Films Doped with Lipophilic Cu(II)-Complex: A Potential Route to the Preparation of Thromboresistant Coatings. Biomaterials 2004, 25 (2), 283–293. https://doi.org/10.1016/S0142-9612(03)00530-1.
(3) Khamespanah, F.; Marx, M.; Crochet, D. B.; Pokharel, U. R.; Fronczek, F. R.; Maverick, A. W.; Beller, M. Oxalate Production via Oxidation of Ascorbate Rather than Reduction of Carbon Dioxide. Nat. Commun. 2021, 12 (1), 1997. https://doi.org/10.1038/s41467-021-21817-w.

Acknowledgements: Research reported in this publication was supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number R01HL168099 and R01HL155100-02S1. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Figure 1: Proposed simplified ascorbate decomposition pathway