Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.
Sport and Health Sciences, College of Life and Environmental Sciences, St Luke's Campus, University of Exeter.
J Vis Exp. 2021 Jul 29(173). doi: 10.3791/62427.
Nitrate ions (NO3) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.
硝酸盐离子（NO3）曾被认为是一氧化氮（NO）代谢的惰性终产物。然而，先前的研究表明，硝酸盐离子可以通过两步还原机制在哺乳动物中被转化回 NO：硝酸盐主要由口腔共生菌还原为亚硝酸盐（NO2），然后通过几种机制（包括通过含铁或含钼的蛋白质）将亚硝酸盐还原为 NO。这种还原型硝酸盐途径有助于增强 NO 介导的信号通路，特别是在心血管系统和肌肉运动期间。体内硝酸盐的水平取决于两个不同的来源：内源性 NO 氧化和主要来自植物的饮食硝酸盐摄入。为了阐明生理情况下复杂的 NO 循环，我们进一步研究了其代谢物硝酸盐和亚硝酸盐离子的动态，与 NO 相比，它们相对稳定。在以前的研究中，骨骼肌被确定为哺乳动物中硝酸盐离子的主要储存器官，也是运动期间 NO 的直接来源。因此，建立一种可靠的方法来测量骨骼肌中的硝酸盐和亚硝酸盐水平非常重要，并且应该有助于将其应用于其他组织样本。本文详细说明了使用三种不同的匀浆方法来制备骨骼肌样品以进行硝酸盐和亚硝酸盐测量的过程，并讨论了与匀浆过程相关的重要问题，包括样品的大小。还比较了四个不同肌肉群中的硝酸盐和亚硝酸盐浓度。
