作者要感谢在制定血液中的亚硝酸盐测量使用亚硝酸盐保存液A. Dejam博士和MM佩尔蒂埃的重要贡献。

所有协议，包括使用动物都是由NIDDK动物护理和使用委员会批准使用，并从美国国立卫生研究院血库健康供者获得人血。 1.样品制备<ol><li> 亚硝酸盐保存溶液的制备 <ol><li>制备含890毫铁氰化钾溶液（K 3的Fe（CN）6）和118毫NEM（N-乙基马来酰亚胺）在蒸馏水。充分溶解，直到很明显的黄色，没有晶体存在。 9比例（V / V，NP-40 /溶液）:在一个1添加的NP-40（辛基phenoxylpolyethoxylethanol）。轻轻混匀，避免起泡，并储存在4℃下一个星期左右）。 </li></ol></li><li> 收集和制备的血液样品的 <ol><li>使用至少20G的针，以避免与肝素溶血防止凝血（5U / ml）的收集血液。用于全血样品，与亚硝酸盐在1立即保存液混合血液:4的比例（ⅴ/ v溶液/血）。 </li><li>对于血浆和红血细胞的样品，离心在4℃下将收集的血液在4000 xg离心5分钟以分离红细胞（RBC）和血浆。取上清（血浆），并将其与上述用于测定血浆中的亚硝酸盐水平的所述亚硝酸盐保存液混合。 </li><li>小心除去残留从管的底部的样本和吸移管红细胞样品的血浆和血沉棕黄层，以避免由其它类型的使用的截止枪头血细胞的污染，并转移到一个管含有在同一亚硝酸保存液比如上述。 </li><li>混合各样品（血浆和红细胞）用冷甲醇在一个比为1:1或1:2（V / V样品/甲醇）和以13,000 xg离心离心他们在4℃15分钟以沉淀蛋白质。取上清，并使用它用于亚硝酸盐测量或冻结制备的样品，如果需要的话，在干冰上并储存在-80℃。 注:亚硝酸盐快速REA克拉与血液形成硝酸盐氧合血红蛋白（oxyHb）。因为oxyHb总是存在于大过量的亚硝酸盐，该反应导致具有的〜10分钟的时间帧天然血液亚硝酸盐的几乎完全湮没。为了保存最内源性血液亚硝酸盐，亚硝酸盐，防腐剂溶液在抽血后立即加入到全血样品。该解决方案将快速溶解红血细胞和氧化oxyHb成高铁血红蛋白（metHb），血红蛋白的无活性形式，不发生化学亚硝酸盐反应。 </li></ol></li><li> 样品从其它类型的体液的制备 <ol><li>样品收集到适当的容器后，用亚硝酸盐保存液，deproteinate拌匀，即可测量或在-80℃冷冻和储存。 </li></ol></li><li> 收集和制备的组织样本 <ol><li>从收集与肝素生理盐水（10U HEPA灌注动物组织RIN / ml）中。消费约1g所需的组织的，均质或者使用手动匀浆器或自动均化器。 </li><li>添加亚硝酸盐根据需要实现平滑匀浆保存液的已知量。 </li><li>如上所述（:1或1 2比V / V样品/甲醇1），以13,000 xg离心然后离心样品在4℃，15分钟一次组织匀浆化，用冷甲醇沉淀蛋白质。 </li><li>收集上清和测量亚硝酸盐的含量。如果需要的话，在制备干冰和存储的任何阶段冷冻样品在-80℃下。 </li></ol></li></ol> 2.减少溶液的制备<ol><li> 三碘（I 3）减少亚硝酸盐和R-（X）-NO种决心1,3,4解决方案 <ol><li>用138在水中毫I 2溶液一起制备301毫碘化钾的溶液。 7比上约30分钟，直到所有的磁力搅拌器（体积/体积溶液/乙酸）:在2冰醋酸混合晶体溶解。最好是保持在黑暗瓶，碘化物对光敏感，并准备在一周内使用。 注意:此解决方案将减少亚硝酸盐成NO，它也将自R-SNO，R-NNO和Fe-NO官能团（R-（X）-NO）释放NO，但硝酸也不会受到影响。从上面的基于无官能基的信号可以从真亚硝酸信号通过用酸化的磺胺（AS）和进行比较的AS-处理和未处理的信号的样本的一半隔开。 AS不可逆地形成重氮阳离子与亚硝酸并且不能由我3溶液可以减少这种复杂。对于AS处理，制备磺胺290毫溶液在1M HCl和它在比1添加到样品的等分试样:9（V / V AS /样品）。测量在样品的AS-处理和未处理部分CL信号。计算真实的亚硝酸盐的信号作为样本的AS-处理的和未处理部分的差。亚硝酸盐可以通过使用选择性尼特可以还测定RITE还原为部分2.2描述抗坏血酸/乙酸混合物溶液。但是，由于R-（X）-NO品种目前的通常少量使用AS-未经处理的样品测量是在大多数情况下，总的亚硝酸盐含量的一个很好的近似。 </li></ol></li><li> 抗坏血酸/乙酸（4A）为亚硝酸盐2的选择性测定溶液 <ol><li>制备在水中的500mM抗坏血酸。混合用冰醋酸该溶液在一个比为1:7（体积/体积，抗坏血酸/乙酸）中以制备反应混合物。 注意:此溶液是具体的亚硝酸盐，不会从任何R-（X）-NO物种或硝酸盐释放NO。然而，抗坏血酸和醋酸的溶液必须被新鲜的测量之前每天制备，如抗坏血酸容易在溶液中氧化。 亚硝酸盐还原的完成取决于抗坏血酸的浓度;并建议完全r至少为50 mm抗坏血酸血浆中的亚硝酸盐排出。然而，建议与最终样品测量之前对不同浓度的抗坏血酸和亚硝酸盐的标准在预期亚硝酸浓度范围执行一些预试验。请记住，抗坏血酸在测量过程中略有消耗，建议在玻璃反应室的反应混合物如此频繁的变化。 </li></ol></li><li> 钒（III）硝酸盐测定氯9减少解决方案 <ol><li>制备的VCl 3的1 M盐酸51毫溶液。通过200微米的过滤器和储存在一个黑暗的瓶过滤器解决方案，在两个星期内使用。 注意:此解决方案将减少硝酸盐，亚硝酸盐和所有R-（X）-NO物种，因此，如果比较的量的亚硝酸盐或其它R-（X）的-NO物种存在与硝酸一起，最终硝酸盐内容来计算与的VCl 3和I中得到的CL信号之间的差3降低了解决方案。 </li></ol></li></ol> 3.化学发光（CL）分析仪的安装和测量<ol><li> 标准的解决方案 <ol><li>制备的亚硝酸钠1μM的溶液（ 亚硝酸钠）。相同的溶液可用于亚硝酸盐和硝酸盐测定。 </li></ol></li><li> 不使用分析仪 注意:目前有两种市售是用于生物研究的目的不够敏感NO分析仪-西弗斯NOA和Ecophysics CLD 88Y。他们都同样的原则进行操作;的主要区别在于，CLD 88Y使用氧气从室内空气，使臭氧（O 3），而NOA要求用于此目的的外部氧气罐。下面的过程说明了设立西弗斯NOA。 <ol><li> 仪器设置 <ol><li>根据制造商的建议进行NO分析仪的初始设置如看到的图1。 </li><li>打开O 2罐连接到仪器。选择"分析"，并从面板上的主菜单中的"输入"。在下一屏幕上选择"开始"，然后按"Enter"键。 （含有1 M氢氧化钠）酸阱连接到仪器， 如图1中所见。 </li><li>等到光电倍增冷却至温度低于-12℃，将反应室抽真空至6乇。大约需要30分钟才能达到稳定平稳的基线。基线需要1内稳定 - 2毫伏，其标称值比其稳定性不太重要。 注意:如果硝酸盐测量，打开冷却水浴（设定为4℃，作为冷阱为酸和水蒸汽）和加热水浴（主反应室中，95℃）。的硝酸盐还原成否的VCl 3溶液的速率是温度相关的，并且它在室温坦佩非常缓慢叉涂抹，需要温度，所以大幅度提高，观察反应。 </li><li>打开他坦克和玻璃反应室连接到酸陷阱在图1中所看到。填充适当还原溶液的反应室，减少鼓泡到最小和反应室连接到酸陷阱。采用试错，调整他冒泡率匹配仪器吸率（对西瑞尔的NOA通常〜200毫升/分钟）。 </li><li>打开电脑控制仪器并启动基于LabVIEW的Liquid软件。要打开仪器和采集软件，按之间的通信"进入"中的"数据菜单"，直到屏幕上显示"输出使能"的时候，然后按"清除"要返回数据的屏幕。 </li><li>等待屏幕上的一个稳定的基线跟踪并开始注入样本和亚硝酸盐标准纳入通过隔膜还原溶液。总是等到达到稳定的基线船尾呃高峰。这最多需要2分钟。液体的软件有一个选项为"标记"时代注入和注释注入，它将这些意见导出到一个单独的文件（filename.info）作为一个有益的补充数据文件（filename.data）。 </li><li>一旦样品和标准衡量，选择"停止"在Liquid软件菜单选项 - 这会从临时文件中的数据写入到被称为"filename.data"永久的文件。按"退出"选项，将终止测量没有获得的数据写入到一个永久文件。 </li><li>终止实验，通过在反应容器的顶部关闭旋塞从反应容器断开机器并断开酸阱和反应容器之间的管（ 见图1）。现在停止NOA分析 - 按"清除"，进入"分析"，把机器上的"待机"和"确认"待命。如果经常，使用的仪器可在待机模式下放置数周。关闭氧气供应。然后冲洗从反应室，断开的还原性溶液，并关闭从玻璃反应容器中，他罐和完成清洁反应容器中。 </li></ol></li><li> 标准和样品进样 <ol><li>注入50微升的1μM的亚硝酸盐溶液成使用公洗涤Hamilton注射器反应混合物。等待至少1 - 注射之间2分钟达到高峰的良好分离。 50微升重复注射以获得复制到每个数据点。每次注射后要用去离子水的注射器。 </li><li>为了获得全套的标准曲线数据，继续与100，150微升重复注射1微米的亚硝酸盐溶液（如果预期的高浓度的亚硝酸盐或硝酸盐可能需要额外的200微升）。在每种情况下，照顾等到信号回落至基线，然后获得1 - 2英里基准的n个达到峰值的良好分离。 注意:亚硝酸盐的标准可在实验过程中的任何时间进行测量。然而，优选的是采集数据测量前的标准曲线，因为它也可作为仪器的功能的独立的检查。 当收集数据时，样品的注入量应导致良好明显的峰值。然而，应当记住的是，所述光电倍增管是线性的仅达〜800毫伏，所以没有峰应比〜700毫伏以上。这可以通过注入量样品的降低或用去离子水稀释的样品来实现任一。每个点应该至少一式两份进行测定。 </li></ol></li></ol></li></ol>

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艾伦·谢克特博士被列为在发给美国国立卫生研究院的使用亚硝酸盐对心血管疾病的治疗几项专利的共同发明人。他接收基于这些专利用于临床开发，但没有其他补偿NIH许可费。

该议定书中的关键步骤用于制备，稀释或以其他方式处理原始样品的所有解决方案（包括水）的等分试样必须保存并检查可能的亚硝酸盐或（更通常）硝酸盐的污染。我们发现大多数污染来自水和用于治疗样品（铁氰化物，特别）许多化学物质也包含在一些地段，与内源性的硝酸盐判定干扰硝酸盐污染的显著量。因此，我们检查所有化学品我们对亚硝酸盐和硝酸盐污染之前，我...

亚硝酸盐，并不太延长硝酸盐，血中水平反映机体的整体状态NO代谢。在血液亚硝酸盐的浓度和最器官和组织是仅在高纳摩尔或低微摩尔范围，硝酸盐通常存在高得多的量 - 在微摩尔范围内。在亚硝酸盐水平由于疾病进展变化或饮食习惯的改变是相当小的，并且可以使用一个非常敏感的方法只测定。由于它们非常不同等级和不同的代谢过程，硝酸盐和亚硝酸盐水平的单独的测定是必不可少的。所谓哪里硝酸盐和亚硝酸盐是在一起测量具有非常小的值"NO x的决心"。 关于各种生物样品中定量亚硝酸盐的几种方法已经被开发 - 最常见的是最老的，基于已在1879年即使现代改性中最初描述的格里斯反应NS，亚硝酸盐达到由格里斯&#39;方法的灵敏度极限是低微摩尔范围内。化学发光（CL）与三碘化物还原溶液相结合，被认为是当前最敏感的方法，允许量化在亚硝酸盐的浓度1-8,10,11的低纳摩尔范围内。相同的CL的方法，用三氯化钒还原溶液相结合，可以用于硝酸盐敏感测量，精确在纳摩尔范围9。 CL检测游离气体NO。因此，亚硝酸盐，硝酸盐，R-亚硝基硫醇（R-SNO），R-亚硝胺（R-NNO），或金属-NO化合物（以后在手稿称作"R-（X）-NO"），必须将其转换成免费无气，以通过CL量化其原始金额。转化为NO是使用几种不同的减少解决方案来实现，这取决于NO代谢物的性质。转换后，游离NO的气体从反应容器中由载体气体（他，N-吹扫2或Ar）到CL分析仪的反应室，其中臭氧（O 3）与NO组合以形成二氧化氮（NO 2）在它的激活状态。与返回到基态，NO 2 *发射在红外线区域和发射的光子由CL仪的光电倍增管（PMT）来检测。发出的光的强度成正比的反应室的NO浓度，这允许使用适当的校准曲线原种的浓度的计算。 在我们的协议，我们硝酸盐和亚硝酸盐的第一本基于CL-测定中最常用的临床设置 - 在血液和血浆，然后我们将讨论如何确定组织样本中的这些离子。我们还详细解释如何保持在亚硝酸盐反应的环境中，如血液及其隔间，血浆和红血细胞的原始生理亚硝酸盐浓度。

<table><tbody><tr><td>potassium ferricyanide; K&lt;sub&gt;3&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma</td><td>702587</td><td></td></tr><tr><td>NEM; N-ethylmaleimide</td><td>Sigma</td><td>4260</td><td></td></tr><tr><td>NP-40; 4-Nonylphenyl-polyethylene glycol</td><td>Sigma</td><td>74385</td><td></td></tr><tr><td>sulfanilamide; AS&amp;nbsp;</td><td>Sigma</td><td>S9251</td><td></td></tr><tr><td>HCl</td><td>Sigma</td><td>H1758</td><td></td></tr><tr><td>acetic acid, glacial</td><td>Sigma</td><td>A9967</td><td></td></tr><tr><td>ascorbic acid&amp;nbsp;</td><td>Sigma</td><td>A7506</td><td></td></tr><tr><td>potassium iodide; KI</td><td>Sigma</td><td>60399</td><td></td></tr><tr><td>iodine; I&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>207772</td><td>light sensitive, toxic</td></tr><tr><td>sodium nitrite; NaNO&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>563218</td><td></td></tr><tr><td>vanadium(III) chloride; VCl&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>208272</td><td>ligt sensitive, toxic</td></tr><tr><td>GentleMac</td><td>Miltenyi</td><td></td><td></td></tr><tr><td>Sievers NOA 280i</td><td>GE</td><td></td><td></td></tr><tr><td>CLD 88Y</td><td>&amp;nbsp;Ecophysics&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

measuring nitrite and nitrate, metabolites in the nitric oxide pathway, in biological materials using the chemiluminescence method

一氧化氮（NO）是在血管内平衡，并且也是神经递质主要调节分子之一。酶产生的NO是由各种含氧血红素蛋白等仍然不为人所熟知的途径相互作用氧化成亚硝酸盐和硝酸盐。相反的过程，还原硝酸盐和亚硝酸盐的成NO已经在哺乳动物中发现了在过去十年中，它是受到关注的可能的途径，以防止或减轻，被认为是心血管，代谢和肌肉疾病的整个范围中的一个与NO的水平降低有关。血液，体液及各种组织 - 它来估计在不同的身体区室NO及其代谢物的量是重要的。血，因为它容易获得，是用于NO代谢物的估计优选隔室。由于它的使用寿命短（几毫秒）和低次纳摩尔浓度，血液没有直接可靠的测量&lt;EM&gt;在体内存在很大的技术困难。因而NO可用性是根据其氧化产物，亚硝酸盐和硝酸盐的用量通常估计。这两种代谢物始终单独测量。有几种公认的方法来确定在生物体液和组织中的浓度。在这里，我们提出了一个协议，用于化学发光法（CL）的基础上，分别由三碘或三氯化钒的解决方案，亚硝酸盐或硝酸盐还原后分光光度检测NO的。对亚硝酸盐和硝酸盐的检测灵敏度低纳摩尔范围，这台CL作为目前最敏感的方法来确定NO代谢途径的变化。我们详细讲解了如何才能在收集，以及如何的时间来确定样品中相应的金额保留原始金额硝酸盐和亚硝酸盐存在的准备从生物体液和组织样本。在CL技术的局限性也是EXPlained。

图2示出了从标准和五种不同样品收集代表性结果。如该图所示，光电倍增电压增加溶液（标准或样品）含亚硝酸注入还原溶液后立即返回到基线值的所有出现在注射一次亚硝酸盐（注射时间由曲线下方红色箭头表示）溶液降低。它也是从该图准确体积的测量是必要的，以获得高重现性的数据清楚。我们建议采用精密汉密尔顿注射器来衡量的注射量。减少I <sub...

Watch this Scientific Journal Video about Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method at JoVE.com

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

用化学发光法测定硝酸盐和亚硝酸盐，代谢产物中的一氧化氮途径，在生物材料

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

measuring-nitrite-nitrate-metabolites-nitric-oxide-pathway-biological

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Unit 1

Signaling Networks of G Protein-coupled Receptors

SCIENTISTS IN ACTION

22.4K 次观看.  NIDDK, NIH. Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

视频: Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of &#945;-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

&#945;-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

用细胞色素 P450s 检测亚硝胺的体外Nitrosamide 形成的一般方法

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

科研

癌症研究

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

一氧化氮气体防治害虫实验室熏蒸程序

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

环境科学

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

为含量测定一氧化氮生物活性分析技术

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

医学

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

生物发光成像的NADPH氧化酶活性在不同的动物模型

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

免疫与感染

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

在生物环境的新技术的产生和观测化学发光

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

生物工程

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

板基检测血液中一氧化氮的 vasp 磷酸化

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

一氧化氮和超氧化物歧化酶恩的人脸检测在完整的动脉内皮细胞层

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

生物学

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the effects of nitrosated compounds. Measuring NO metabolites is essential to studying NO activity both at vascular levels and in other tissues, especially in the experimental settings where exogenous NO is administered. Ozone-based chemiluminescence assays allow precise measurements of NO and NO metabolites in both fluids (including plasma, tissue homogenates, cell cultures) and gas mixtures (e.g., exhaled breath). NO reacts with ozone to generate nitrogen dioxide in an excited state. The consequent light emission allows photodetection and the generation of an electric signal reflecting the NO content of the sample. Aliquots from the same sample can be used to measure specific NO metabolites, such as nitrate, nitrite, S-nitrosothiols, and iron-nitrosyl complexes. In addition, NO consumed by cell-free hemoglobin is also quantified with chemiluminescence analysis. An illustration of all these techniques is provided.

Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

基于化学发光的检测方法，用于检测自氧化和亚硝化化合物中的一氧化氮及其衍生物

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

遗传编码的荧光一氧化氮的应用（NO•）探针，所述geNOps，用于在单细胞NO•信号的实时影像

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

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.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

用于硝酸盐和亚硝酸盐测量的大鼠骨骼肌匀浆的制备

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...