Name: measuring nitrite and nitrate, metabolites in the nitric oxide pathway, in biological materials using the chemiluminescence method
Uploaded: 2016-12-25T15:00:00
Description: 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

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...

<ol>
	<li>Piknova, B., Schechter, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Nitrite+in+Blood+Samples+Using+the+Ferricyanide-Based+Hemoglobin+Oxidation+Assay.">Measurement of Nitrite in Blood Samples Using the Ferricyanide-Based Hemoglobin Oxidation Assay.</a> Methods Mol Biol. 704, 39-56 (2011).</li><li>Nagababu, E., Rifkind, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+plasma+nitrite+by+chemiluminescence+without+interference+of+S-,+N-nitroso+and+nitrated+species.">Measurement of plasma nitrite by chemiluminescence without interference of S-, N-nitroso and nitrated species.</a> Free Radic Biol Med. 42, 1146-1154 (2007).</li><li>Pinder, A. G., Rogers, S. C., Khalatbari, A., Ingram, T. E., James, P. E., Hancock, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+nitric+oxide+and+its+metabolites+in+biological+samples+by+ozone-based+chemiluminescence.">The measurement of nitric oxide and its metabolites in biological samples by ozone-based chemiluminescence.</a> Methods in Molecular Biology, Redox-Mediated Signal Transduction. 476, 11-28 (2008).</li><li>Pelletier, M. M., Kleinbongard, P., Ring-wood, L., Hito, R., Hunter, C. J., Schechter, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+blood+and+plasma+nitrite+by+chemiluminescence:+pitfalls+and+solutions.">The measurement of blood and plasma nitrite by chemiluminescence: pitfalls and solutions.</a> Free Radic Biol Med. 41, 541-548 (2006).</li><li>Mac Arthur, P. H., Shiva, S., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+circulating+nitrite+and+S-nitrosothiols+by+reductive+chemiluminescence.">Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence.</a> J Chromatogr B. 851, 93-105 (2007).</li><li>Bryan, N. S., Grisham, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+detect+nitric+oxide+and+its+metabolites+in+biological+samples.">Methods to detect nitric oxide and its metabolites in biological samples.</a> Free Radic Biol Med. 43, 645-657 (2007).</li><li>Hendgen-Cotta, U., Grau, M., Rasaaf, T., Gharinin, P., Kelm, M., Kleinbongard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductive+gas-phase+chemiluminescence+and+flow+injection+analysis+for+measurement+of+nitric+oxide+pool+in+biological+matrices.">Reductive gas-phase chemiluminescence and flow injection analysis for measurement of nitric oxide pool in biological matrices.</a> Method Enzymol. 441, 295-315 (2008).</li><li>Yang, B. K., Vivas, E. X., Reiter, C. D., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+for+the+sensitive+and+specific+measurement+of+S-nitrosothiols,+iron-nitrosyls+and+Nitrite+in+biological+samples.">Methodologies for the sensitive and specific measurement of S-nitrosothiols, iron-nitrosyls and Nitrite in biological samples.</a> Free Radic Res. 37, 1-10 (2003).</li><li>Sm&#225;rason, A. K. 1., Allman, K. G., Young, D., Redman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+levels+of+serum+nitrate,+a+stable+end+product+of+nitric+oxide,+in+women+with+pre-eclampsia.">Elevated levels of serum nitrate, a stable end product of nitric oxide, in women with pre-eclampsia.</a> Br J Obstet Gynaecol. 104 (5), 538-543 (1997).</li><li>Beckman, J. S., Congert, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Measurement+of+Dilute+Nitric+Oxide+in+Solution+with+an+Ozone+Chemiluminescent+Detector.">Direct Measurement of Dilute Nitric Oxide in Solution with an Ozone Chemiluminescent Detector.</a> Methods: A companion to Methods in Enzymology. 7, 35-39 (1995).</li><li>Bates, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+measurements+by+chemiluminescence+detection.">Nitric oxide measurements by chemiluminescence detection.</a> Neuroprotocols: A companion to Methods in Neuroscience. 1, 141-149 (1992).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="812">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:351px;">potassium ferricyanide; K3Fe(CN)6</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">702587</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEM; N-ethylmaleimide</td>
			<td style="width:135px;">Sigma</td>
			<td>4260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40; 4-Nonylphenyl-polyethylene glycol</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sulfanilamide; AS&#160;</td>
			<td style="width:135px;">Sigma</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">HCl</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">H1758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">acetic acid, glacial</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">A9967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ascorbic acid&#160;</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">A7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">potassium iodide; KI</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">60399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">iodine; I2</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">207772</td>
			<td>light sensitive, toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">sodium nitrite; NaNO2</td>
			<td style="width:135px;">Sigma</td>
			<td>563218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">vanadium(III) chloride; VCl3</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">208272</td>
			<td>ligt sensitive, toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GentleMac</td>
			<td style="width:135px;">Miltenyi</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sievers NOA 280i</td>
			<td style="width:135px;">GE</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CLD 88Y</td>
			<td style="width:135px;">&#160;Ecophysics&#160;</td>
			<td style="width:119px;"></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 ...

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