Department of Plastic and Reconstructive Surgery, Hand Surgery, and Burn Center, Medical Faculty, RWTH Aachen University, Pauwelsstr. 30, D-52074 Aachen, Germany.
Nitric Oxide. 2013 Jan 15;28:24-32. doi: 10.1016/j.niox.2012.09.003. Epub 2012 Oct 2.
Topical application of nitric oxide (NO) has been shown to exert beneficial effects in the therapy of chronic wounds, impaired microcirculation, and skin infections. Nitrite acidified by ascorbic acid has been widely used in many studies as NO-donor system, unfortunately with inflammatory and toxic effects on the treated skin due to unregulated excessive NO generation, low pH and possible toxic side products. Here we describe an essentially modified nitrite based NO generating system that avoid the mentioned unwanted side effects on human skin by using a pH-stable acetate/acetic acid buffer with a skin neutral pH of 5.5 and sodium ascorbate. In order to overcome the shortcoming of lower NO yields due to the higher pH-value and low nitrite concentrations, we have determined additionally the influence of copper ions. To investigate the influence of different NO release and penetration kinetics on NO-induced toxicity, we have developed a fibroblast assay using cell culture plates with gas permeable bottoms. The results show clearly that the donor system can achieve a sustained NO generation without generating high peaks. Furthermore, the presence of Cu(2+) ions enhances manifold NO generation of pH/ascorbate-induced nitrite decomposition, a mechanism comprising the reduction of Cu(2+) ions to Cu(1+) by ascorbate. Finally, we have found that apart from the NO dose the NO release kinetics had a significant influence of cell toxicity. Thus, application of comparable NO amounts within a time interval of 600s led to the development of variable cell toxicities, which predominantly depended on the NO concentration values generated in the first 200s. In summary, we here describe a novel nitrite-based NO-donor system that can provide well defined NO concentrations at skin neutral pH-values for side effect poor topical dermal application, i.e. in the therapy of chronic wounds and impaired microcirculation.
局部应用一氧化氮（NO）已被证明对慢性伤口、微循环受损和皮肤感染的治疗具有有益作用。用抗坏血酸酸化的亚硝酸盐已被广泛用于许多研究中作为 NO 供体系统，但由于不受调节的过量 NO 生成、低 pH 值和可能的毒性副产物，对治疗皮肤会产生炎症和毒性作用。在这里，我们描述了一种基本改良的基于亚硝酸盐的 NO 生成系统，该系统通过使用 pH 稳定的醋酸盐/乙酸缓冲液（皮肤 pH 值为 5.5）和抗坏血酸钠来避免对人皮肤产生上述不良副作用，该缓冲液的 pH 值稳定，且呈中性。为了克服由于较高的 pH 值和较低的亚硝酸盐浓度导致的 NO 产率较低的缺点，我们还确定了铜离子的影响。为了研究不同的 NO 释放和渗透动力学对 NO 诱导的毒性的影响，我们使用具有透气底部的细胞培养板开发了一种成纤维细胞测定法。结果清楚地表明，该供体系统可以实现持续的 NO 生成，而不会产生高峰。此外，铜离子的存在大大增强了 pH/抗坏血酸诱导的亚硝酸盐分解产生的 NO 生成，该机制包括抗坏血酸将 Cu(2+) 离子还原为 Cu(1+)。最后，我们发现除了 NO 剂量外，NO 释放动力学对细胞毒性也有显著影响。因此，在 600s 的时间间隔内应用相当量的 NO 会导致细胞毒性产生不同程度的发展，这主要取决于在前 200s 中生成的 NO 浓度值。总之，我们在这里描述了一种新型的基于亚硝酸盐的 NO 供体系统，该系统可以在皮肤中性 pH 值下提供定义明确的 NO 浓度，用于副作用较小的局部皮肤应用，即慢性伤口和微循环受损的治疗。
