Piknova Barbora, Park Ji Won, Cassel Katelyn S, Gilliard Cameron N, Schechter Alan N
Molecular Medicine Branch, NIDDK, NIH;
Molecular Medicine Branch, NIDDK, NIH.
J Vis Exp. 2016 Dec 25(118):54879. doi: 10.3791/54879.
Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments - blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.
一氧化氮（NO）是血管稳态中的主要调节分子之一，也是一种神经递质。通过酶促产生的NO会与各种氧合血红蛋白及其他尚不明确的途径相互作用，被氧化为亚硝酸盐和硝酸盐。在过去十年中，哺乳动物体内发现了亚硝酸盐和硝酸盐还原为NO的逆过程，作为预防或缓解一系列被认为与NO水平降低相关的心血管、代谢和肌肉疾病的可能途径之一，这一过程正受到越来越多的关注。因此，估计不同身体部位（血液、体液和各种组织）中NO及其代谢产物的含量非常重要。血液因其易于获取，是用于估计NO代谢产物的首选部位。由于其寿命短（几毫秒）且亚纳摩尔浓度低，体内直接可靠地测量血液中的NO存在很大的技术困难。因此，通常根据其氧化产物亚硝酸盐和硝酸盐的量来估计NO的可用性。这两种代谢产物总是分别测量。有几种成熟的方法可用于测定它们在生物体液和组织中的浓度。在此，我们介绍一种化学发光法（CL）的方案，该方法分别基于用三碘化物或氯化钒（III）溶液将亚硝酸盐或硝酸盐还原后对NO的分光光度检测。亚硝酸盐和硝酸盐检测的灵敏度处于低纳摩尔范围，这使CL成为目前可用于确定NO代谢途径变化的最灵敏方法。我们详细解释了如何从生物体液和组织中制备样品，以保留采集时存在的亚硝酸盐和硝酸盐的原始量，以及如何确定样品中它们各自的量。还解释了CL技术的局限性。
