(T1130-12-81) Lipophilic Copper Complex for Local Controlled Release of Nitric Oxide from Circulating Nitrites and S-nitrosothiols

Purpose: Nitric oxide (NO) is a small diatomic free radical that plays a prominent role in several physiological functions. NO is crucial to the maintenance of cardiovascular homeostasis, which it does by regulating vascular tone and blood pressure and through its antiplatelet activity. However, free NO radical has a very short half-life in vivo and exists mostly in the form of NO-related species. Nitrite is a NO species of particular interest because it is more stable than free NO and can be converted back to NO by numerous pathways in the body, thus enabling it to function as an NO storage pool. Extracorporeal membrane oxygenation (ECMO) is a method of life support used to provide cardiac and respiratory support for patients unable to independently provide an adequate amount of gas exchange or perfusion themselves. ECMO carries a high chance of clotting that is typically managed with systemic anticoagulants, which then increases the likelihood of bleeding in high-risk patients. The purpose of this study was to investigate the distribution of NO species in blood after NO exposure and to develop a copper containing lipophilic complex to induce the release of NO from nitrite at local sites to prevent thrombus formation.

Methods: For the detection and quantification of nitrite, sulfuric acid (H₂SO₄) and potassium iodide (KI) were used to reduce nitrite to NO, which was then carried in N₂ to the nitric oxide analyzer (NOA) where it was detected through ozone chemiluminescence. Foaming of the samples was mitigated with antifoaming agent Antifoam SE-15. These methods were applied to serum samples. Serum was obtained from a study in which pigs were exposed to 500ppm NO_(g) via membrane oxygenator for two hours during cardiopulmonary bypass surgery. Sheep serum was purchased and then exposed to 50 ppm NO gas via bubbling for two minutes. The lipophilic Cu(II)-complex was synthesized as described previously¹.

Results: Nitrite was quantified in pig serum samples and found to have increased significantly (from 463.47 nM to 5727.7 nM, n=3, p=0.05) during NO exposure. Nitrite was quantified in sheep serum and found to have increased significantly (from 3.218 nM to 376.0 nM, n=3, p=0.05) after NO exposure. Synthesis of the lipophilic copper compound was confirmed with NMR.

Conclusion: The KI and H₂SO₄ assay can be used to determine the concentration of nitrites as a circulating NO reservoir in the blood. Exposure to NO through a membrane oxygenator significantly increases the size of this circulating NO reservoir, and the mechanism of the nitrite formation is not dependent on the presence of red blood cells. Impregnating the tubing used in ECMO circuitry with the lipophilic Cu(II)-complex has the potential to be used in conjunction with NO therapy to locally deliver NO to prevent thrombus formation during ECMO.

References: (1) Oh, B. K.; Meyerhoff, M. E. Spontaneous Catalytic Generation of Nitric Oxide from S-Nitrosothiols at the Surface of Polymer Films Doped with Lipophilic Copper(II) Complex. J. Am. Chem. Soc. 2003, 125 (32), 9552–9553. https://doi.org/10.1021/ja035775x.

Acknowledgements: Research reported in this publication was supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Numbers R01HL155100, 5R01HL141509-04, 5R01EB028775-03, and 1R01HL16809901. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.