Name: a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s
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Description: Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

这项研究得到了格兰特号的支持。CA-81301 来自国家癌症研究所我们感谢鲍勃的编辑协助, Dr. 彼得 Villalta 和鲁迅的质谱协助分析生物化学共享资源共济会癌症中心, 和 Dr. 亚当 t. Zarth 和 Dr. 安娜 k. 米歇尔的宝贵讨论和输入。分析生物化学共享资源由国家癌症研究院癌症中心资助 CA-77598 部分支持

1. 材料和常规过程 <ol> <li> 将 NNN 合成为以前描述的 15 。获得 norcotinine, P450 2A6 Baculosomes, 再生系统, 0.5x 和 #160; 反应缓冲器, 以及试剂级商业来源的所有其他化学品或溶剂. </li>
	<li> 在 500 MHz 光谱仪上记录核磁共振谱。报告化学变化为每百万分之 (ppm)。使用残余溶剂峰值作为内部参考为 1 H-nmr (7.26 ppm CDCl 3 ) 和 13 C-nmr (77.2 ppm CDCl 3 ). 
	注意: 峰值分裂使用了以下简称: s = 单线态, d = 双峰, dd = 双峰的双峰, dt = 三胞胎的双峰, dq = 双峰的四重奏, ddd = 双峰的双峰, 和 m = multiplet. </li>
	<li> 对 LTQ Orbitrap Velos 上所选化合物执行高分辨率质谱 (人力资源), 并将数据报告为 m/z . </li>
	<li> 使用金预硅胶薄层色谱片 (40 毫米 x 80 毫米, 0.2 毫米厚) 与 254 nm 荧光指示剂 (tlc)。通过 UV 灯照射来可视化薄层色板. </li>
	<li> 在60和 #197 上执行闪光色谱; 70-150 目硅胶使用 1.5 cm x 15 cm 玻璃柱. </li>
</ol> 2。nitrosamide 阳性控制 ( N & #8242; -nitrosonorcotinine, NNC) <ol> <li> 干燥, 在烤箱过夜 (16 小时, 〜140和 #176; C), 一个25毫升, 圆底烧瓶含有磁性搅拌棒。早上, 在 n 2 流下冷却烧瓶, 同时使用油起泡使 n 2 流速保持在每秒一个气泡下. 
	注: 冷却后, 不需要在 N 2 下保持烧瓶的预防措施. </li>
	<li> 在通风罩中添加 norcotinine (31.8 毫克、0.196 摩尔)、醋酸酐 (5 毫升) 和乙酸 (1 毫升) 到冷却烧瓶中。在冷却到0和 #176 时, 将此混合物连续搅拌; C 冰浴. 
	 警告: 醋酸酐和乙酸具有腐蚀性. </li>
	<li> 添加纳米 2 (33.3 毫克, 0.483 摩尔) 在一个部分和连续搅拌的混合物为2.5 小时. 监测反应进展的 TLC (100% EtOAc, R f = 0.19, 参见步骤 1.4). 
	注: 在这段时间, 混合物将变得越来越黄与偶尔起泡. </li>
	<li> 通过将混合物倒入 ice-cold H 2 O (18 毫升) 来淬火反应。在 100 ml 分漏斗中, 立即用18毫升 CH 2 Cl 2 提取水溶液。用 CH 2 Cl 2 (9 毫升) 将水层至少再抽出2次. </li>
	<li> 干燥100毫克 MgSO 的混合有机物 4 用于2分钟的过滤, 并通过旋转蒸发浓缩溶液以产生原油、黄油。在蒸发过程中, 将水浴加热到30和 #176; C. 
	注意: 如果化合物在 CH 2 Cl 2 中被溶解, 并且该解决方案在黑暗中存储在 2-8 和 #176 中, 则可以暂停该协议; C. </li>
	<li> 用硅胶作为固定相和 100% EtOAc 为淋洗 16 , 通过柱层析 (见步骤 1.5) 净化粗化合物. 
	 警告: NNC 和相关的 nitrosamides 应小心处理, 因为它们被认为是人类致癌物. </li>
	<li> 溶解 CDCl 中的纯化合物 3 并使用 NMR (参见步骤 1.2) 确认结构并确定此解决方案的浓度 17 。将此解决方案存储在 2-8 和 #176 的黑暗中, 直到需要为止. 
	注意: 此解决方案将用作 体外 检测 (步骤 3.5) 中的正向控制。在支持信息中提供了核磁共振波谱和化学位移. </li>
	<li> 使用纯 NNC 解决方案, 通过在步骤1.3 和4.3 中描述的参数直接注入高分辨率质谱仪 (人力资源), 确定准确的母体质量并测定产品离子质量. 
	注: 产品质量将用于检测该化合物在 体外 代谢试验 (步骤 4.3). </li>
</ol> 3。一个例子 nitrosamine-P450 体外 孵化 <ol> <li> 删除 P450 2A6 Baculosomes, 生动-再生系统, 和 10 mM NADP + 库存解决方案从-80 和 #176; C 冷冻, 让他们解冻冰。一旦解冻, 稀释 Baculosomes 和再生系统1:10 和1:50 分别, 成一个单一的1毫升管与0.5X 反应缓冲器。同样, 添加3和 #956; NADP + 库存解决方案为97和 #956; l 0.5X 反应缓冲器. 
	注意: NADP + 是光敏的。通过将容器包装在铝箔中来保护此解决方案。酶解对冻融循环敏感;将此限制为不超过2个周期。为将来的实验做必要的准备等分. </li>
	<li> 对于每个孵化, 添加50和 #956; 酶溶液 l (含 5 pmol P450) 至1毫升管, 包含40和 #956; l 4 和 #956; M NNN 的解决方案, 由反应缓冲器。预孵化这种新的混合物为2分钟, 在37和 #176; C 使用水浴, 然后添加10和 #956; 稀释的 NADP + 解决方案。孵育整个系统 1-30 分钟, 在37和 #176; C. 
	注: 使用5分钟的潜伏期 (如 5, 10, 15 分钟等) 将提供足够的 nitrosamide 形成时间的过程. </li>
	<li> 在所需的孵育时间后, 先将10和 #956 进行淬火; 3.0 n ZnSO 4 , 然后是10和 #956; 3.0 n Ba (哦) 2 的 l. 漩涡这个解决方案和白色沉淀将形成。离心样品在 8000 x g 为4分钟颗粒的沉淀. 
	注: 此步骤不能暂停, 因为形成的 nitrosamides 在水溶液中的稳定性有限。长时间的停顿可能导致错误的底片. </li>
	<li> 立即将上清液移出, 并分析2和 #956; 用液体色谱法正 nanoelectrospray 电离高分辨串联质谱法 (LC-NSI + -人力资源/毫秒, 步骤 4.1-4.3). </li>
	<li> 对于正控制孵化, 将含有 100 fmol 的合成 NNC 溶液的分蒸发到1毫升的管下, 在 N 的流下为 2 。对这个小瓶, 添加完整的酶系统从步骤3.2 和继续按照描述的步骤 3.3-3.4. </li>
	<li> 对于负控制孵化, 执行所描述的实验 (步骤 3.2-3.4), 除替换50和 #956; L 酶溶液与0.5X 反应缓冲器. </li>
</ol> 4。用于 lc 组件的 lc-NSI + -人力资源/MS <ol> <li> 的 nitrosamide 检测参数的示例, 请将步骤4.2 中的以下多级渐变应用于使用商用、手动包装的 18 C18 (5 和 #956; m) 的 UPLC 系统,100毫米 x 75 和 #956; m, 15 和 #956; m 孔毛细管柱. </li>
	<li> 使用 5 mM NH 4 华侨作为溶剂 a 和 MeCN 作为溶剂 b, 通过在1和 #956 的 5% b 处运行, 将样品加载到列上; L/分钟从 0-5 分钟, 然后减慢流速到0.3 和 #956; 之后的升/分钟。接下来, 运行从5到 20% b 以上4分钟的渐变, 后跟一个斜坡到 55% b 以上10分钟, 然后 re-equilibration 到 5% b. </li>
	<li> 在 LTQ Orbitrap 上执行 LC-NSI + -人力资源/MS。通过全扫描和 MS 2 碎片监视 NNC。以6万的分辨率进行全扫描, 并以 5 ppm 的质量公差提取准确的母质量 (192.07670)。使用 ms 2 碎片分离父离子 (2.0) 和由碰撞引发的离解 (CID) 与 25 eV 的碰撞能量, 分辨率 1.5万, 和扫描时间30毫秒提取的产品离子的准确质量 NNC ( m/z 192 m/z 134.04739 和 162.07874) 的质量公差为 5 ppm. </li>
</ol>

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作者没有什么可透露的。

阐明亚硝胺的代谢是了解其致癌性的重要组成部分。由于涉及的细胞色素 P450s 和其他代谢酶是多态的, 进一步应用这一知识可能会发现高危个体1,4。新的数据表明, 进一步氧化α-hydroxynitrosamines, 推定主要代谢物的亚硝胺参与 DNA 结合, 以 nitrosamides 是可能的;然而, 这并没有在广泛的基板上进行强有力的测试。我们已经描述了一个协议来确定性氧化亚硝胺的...

N-亚硝胺是一种在饮食、烟草制品和一般环境中发现的大量致癌物质;它们也可以在人体内形成内1。超过 300 N-硝基化合物已经被测试和 #62; 90% 被评估了作为致癌物在动物模型2,3。为了展示它们的致癌性, 这些化合物必须首先激活细胞色素 P450s1,2,3。研究表明, 细胞色素 P450s 容易氧化亚硝胺为α-hydroxynitrosamines (图 1), 这是高度活性化合物与半衰期的〜 5 s 之前自发分解到 alkyldiazohydroxides。后者可以在丢失 H2O 和 N2之后烷基化 DNA。由此产生的 DNA 加, 如果未, 可以导致突变, 如果在关键的 onco 或抑癌基因, 导致癌症的发展1。因此, 为了充分了解细胞色素 P450 氧化致癌亚硝胺的代谢途径、DNA 加和下游代谢产物, 已经花费了大量的精力。这些知识在个人癌症风险评估中具有潜在的应用价值4。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56312/56312fig1.jpg"> 
图 1: 一般和建议的亚硝胺代谢。 亚硝胺 (1) 被 P450s 氧化为α-hydroxynitrosamines (2), 自发分解为 alkyldiazohydroxides (3)。这些化合物可以与 dna 结合形成 dna 加。假设2被 P450s 进一步氧化为 nitrosamides 4。这些可以直接绑定到 dna, 形成新的 dna 加或水解到3 , 形成已知的 dna 加。r1和 r2表示任何烷基组。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56312/56312fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
虽然α-hydroxynitrosamine 假说得到了大量数据的坚实支持, 但也存在一些不一致之处。主要的一个是α-hydroxynitrosamines 的短半衰期5,6。众所周知, 这些化合物是在内质网膜和后来的烷基化核 DNA 中产生的。鉴于他们的生命的几秒钟, 这是令人费解的这些中间体如何生存所需的旅行, 虽然胞。一个假说是, α-hydroxynitrosamines 的一部分被 processively 氧化为 nitrosamides7,8, 在比较9中相当稳定。这可能会发生通过保留α-hydroxynitrosamines 在细胞色素 P450 活性部位。这种类型的氧化的先例已经看到与尼古丁10, 酒精的11, 和简单的 alkylnitrosamines12,13。另外, nitrosamides 是直接作用的致癌物质2,3。根据它们的反应性9, 这些化合物被认为产生的 dna 加与α-hydroxynitrosamines 和新的, 未经探索的 dna 加 (图 1) 的结果相同。因此, 这一假说不仅解释了通过胞的运输, 也说明了 DNA 损伤产物的形成。
本文介绍了一种用于评价亚硝胺的细胞色素 P450-mediated 转化为 nitrosamides 的体外的通用协议。先前报告的′-nitrosonornicotine (NNN) 转换为′-nitrosonorcotinine (NNC) 的细胞色素 P450 2A6 被展示为示例14。该协议在广泛的基质酶系统的应用将有助于确定 nitrosamides 在整体硝代谢中的重要性。

<table><tbody><tr><td>Norcotinine</td><td>AKoS GmbH (Steinen, Germany)</td><td>CAS 17708-87-1, AKoS AK0S006278969</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>695092</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Sigma-Aldrich</td><td>242845</td><td></td></tr><tr><td>Ammonium Acetate</td><td>Sigma-Aldrich</td><td>431311</td><td></td></tr><tr><td>Barium Hydroxide</td><td>Sigma-Aldrich</td><td>433373</td><td></td></tr><tr><td>D-Chloroform</td><td>Sigma-Aldrich</td><td>151823</td><td></td></tr><tr><td>HPLC Acetonitrile</td><td>Sigma-Aldrich</td><td>34998</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Methylene Chloride</td><td>Sigma-Aldrich</td><td>34856</td><td></td></tr><tr><td>Sodium Nitrite</td><td>Sigma-Aldrich</td><td>237213</td><td></td></tr><tr><td>ViVid CYP2A6 Blue Screening Kit</td><td>Life Technologies</td><td>PV6140</td><td></td></tr><tr><td>Zinc Sulfate</td><td>Sigma-Aldrich</td><td>221376</td><td></td></tr><tr><td>0.5 mL tubes</td><td>Fisher</td><td>AB0533</td><td></td></tr><tr><td>100 mL round bottom flask</td><td>Sigma-Aldrich</td><td>Z510424</td><td></td></tr><tr><td>125 mL Erlenmeyer flask</td><td>Sigma-Aldrich</td><td>CLS4980125</td><td></td></tr><tr><td>125 mL Separatory Funnel</td><td>Sigma-Aldrich</td><td>Z261017</td><td></td></tr><tr><td>25 mL round bottom flask</td><td>Sigma-Aldrich</td><td>Z278262</td><td></td></tr><tr><td>500 MHz NMR Spectrometer</td><td>Bruker</td><td></td><td></td></tr><tr><td>Allegra X-22R Centrifuge</td><td>Beckman-Coulter</td><td></td><td></td></tr><tr><td>LC vials</td><td>ChromTech</td><td>CTC&amp;ndash;0957&amp;ndash;BOND</td><td></td></tr><tr><td>LTQ Orbitrap Velos</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Magnetic Stir bar</td><td>Sigma-Aldrich</td><td>Z127035</td><td></td></tr><tr><td>NMR tube</td><td>Sigma-Aldrich</td><td>Z274682</td><td></td></tr><tr><td>P1000, P200, and P10 pipettes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Rotary evaporator</td><td>Sigma-Aldrich</td><td>Z691410</td><td></td></tr><tr><td>RSLCnano UPLC system</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Shaking Water Bath</td><td>Fisher</td><td>FSSWB15</td><td></td></tr><tr><td>Stir plate</td><td>Sigma-Aldrich</td><td>CLS6795420</td><td></td></tr><tr><td>PicoFrit Column</td><td>New Objective</td><td>PF3607515N5</td><td></td></tr><tr><td>Luna C18, 5 um</td><td>Phenomenex</td><td>535913-1</td><td></td></tr></tbody></table>

a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s

N-亚硝胺是一种公认的环境致癌物质, 需要细胞色素 P450 氧化来表现活性。代谢活化机制包括自发分解为 DNA 烷剂的α-hydroxynitrosamines 的形成。DNA 损伤的积累和由此产生的突变最终会导致癌症。新的证据表明, α-hydroxynitrosamines 可以进一步氧化到 nitrosamides processively 细胞色素 P450s。因为 nitrosamides 一般比α-hydroxynitrosamines 更稳定, 也可以烷基化 DNA, nitrosamides 可能在癌变中起到作用。在本报告中, 我们描述了一个一般的协议, 评估 nitrosamide 生产从体外细胞色素 P450-catalyzed 代谢亚硝胺。该协议采用了一般的方法合成相关的 nitrosamides 和一个体外细胞色素 P450 代谢测定的液相色谱-nanospray 电离-高分辨率串联质谱检测。此方法在示例研究中检测到′-nitrosonorcotinine 作为′nitrosonornicotine 的次要代谢物。该方法具有较高的灵敏度和选择性, 是由于质量检测准确。该方法在多种硝细胞色素 P450 系统中的应用将有助于确定这种转化的普遍性。由于细胞色素 P450s 的多态性和活动性的变化, 更好地了解 nitrosamide 的形成有助于个别癌症风险评估。

基于白色et al的工作。19, norcotinine 是硝化 NNC 清洁和高产 (80-92%), 以产生一个标准的体外实验。一个成功的反应的结构证据获得了从光谱分析包括1H 核磁共振, 13C 核磁共振, 舒适, 和 HSQC (支持信息) 与人力资源证实了父质量 [M + h]+在 5 ppm 的理论值 (图 2)。MS2的碎片 NNC 被用来确定最丰富的?...

Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

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

细胞色素 P450s 致癌亚硝胺的α-羟基化是公认的代谢途径, 产生 DNA 损伤的中间体, 导致突变。然而, 新的数据表明进一步的氧化 nitrosamides 可能发生。我们描述了一个一般的方法, 检测 nitrosamides 产生的体外细胞色素 P450-catalyzed 代谢的亚硝胺。

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 ...

a-general-method-for-detecting-nitrosamide-formation-vitro-metabolism

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

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

10.0K 次观看.  University of Minnesota. 细胞色素 P450s 致癌亚硝胺的α-羟基化是公认的代谢途径, 产生 DNA 损伤的中间体, 导致突变。然而, 新的数据表明进一步的氧化 nitrosamides 可能发生。我们描述了一个一般的方法, 检测 nitrosamides 产生的体外细胞色素 P450-catalyzed 代谢的亚硝胺。

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

质谱法和基于发光的-途径表征阶段我代谢能力的<em&gt;体外</em&gt;细胞培养

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

生物化学

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其对32 P-postlabeling 的测定

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

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

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

利用高效液相色谱-电化学检测仪检测大气环境中的 3-硝基酪氨酸

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

环境科学

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

微型固相萃取和LC-MS / MS检测人尿中3-硝基酪氨酸的临床应用

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

化学

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO3&#8722; for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO3&#8722; transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the 15N-labeled ammonium (15NH4+) accumulation rate in 15NO3&#8722; added incubation. The determination of the 14NH4+ and 15NH4+ concentrations described in this paper is comprised of the following steps. First, the NH4+ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO3&#8722; via persulfate oxidation. Third, the NO3&#8722; is converted to N2O via an N2O reductase deficient denitrifier. Finally, the converted N2O is analyzed using a previously developed quadrupole gas chromatography&#8211;mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

A series of methods to determine the potential DNRA rate based on 14NH4+/15NH4+ analyses is provided in detail. NH4+ is converted into N2O via several steps and analyzed using quadrupole gas chromatography&#8211;mass spectrometry.

基于14NH 4 + /4+ 15NH 4= 通过顺序转换为 N2O 的分析，测量异化硝酸盐降低至铵的潜在率

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

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 ...

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

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 &#8212; 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.

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 ...

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

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