Name: biochemical measurement of neonatal hypoxia
Uploaded: 2011-08-24T13:00:00
Description: Watch this Scientific Journal Video about Biochemical Measurement of Neonatal Hypoxia at JoVE.com

这项工作是由美国国立健康R01 NR011209 - 03研究院

1。样品的采集与加工<ol><li>收集血液样本，在6毫升K3E EDTA K3管放在冰上。 </li><li>收集2分钟内，离心机的样品在4 ° C时为10分钟的1500克。 </li><li>上清（浆）转移到1.5ml离心管。 </li><li>在4 ° C离心30分钟在18000克。 </li><li>去除上清分装和嘌呤（200μL），尿囊素（50μL），MDA（100μL），和XO（120μl）分析他们转移到不同的离心管。要小心，不要污染与红血细胞的样品。您可能需要调整血浆MDA，XO，并?...

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这里所描述的方法，允许新生儿缺氧缺血的评价。该协议结合的能量（ATP）剥夺，氧化应激，氧化损伤，酶的活性标志物的测量，以获得一个整体的存在生化图片甚至缺氧缺血程度。尽管这种方法的有效性，也有潜在的局限性。首先，它需要大约1-2毫升血液收集到足够的血浆运行的所有实验。这会不会是成人或儿童的问题，但新生儿，特别是那些过早时，它成为一个关注。优先检测和稀释的一些?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> 6毫升K3E EDTA K3管</td><td> Fisher Scientific则</td><td> 2204061 </td><td></td></tr><tr><td> 5702R离心机</td><td> Fisher Scientific则</td><td> 05413319 </td><td> 13＆16MM适配器</td></tr><tr><td> 1.5ml离心管</td><td>美国科学</td><td> 1615-5599 </td><td></td></tr><tr><td> 2 - 氨基嘌呤</td><td> Sigma - Aldrich公司</td><td> A3509 </td><td></td></tr><tr><td>瓦里安卡里100分光光度计</td><td> Agilant技术</td><td> 0010071500 </td><td></td></tr><tr><td> SAVANT Speed​​Vac </td><td> Thermo Scientific的</td><td> SC210A - 115 </td><td></td></tr><tr><td>美光科技公司的离心过滤装置</td><td> Fisher Scientific则</td><td> UFC501596 </td><td></td></tr><tr><td> Supelcosil LC - 18 - S列</td><td> Sigma - Aldrich公司</td><td> 58931 </td><td></td></tr><tr><td> Supelcosil LC - 18 - S Supelguard墨盒和支架</td><td> Sigma - Aldrich公司</td><td> 59629 </td><td></td></tr><tr><td>高效液相色谱法</td><td>沃特斯</td><td></td><td></td></tr><tr><td> GCMS瓶</td><td> Fisher Scientific则</td><td> 03376607 </td><td></td></tr><tr><td> DL -尿囊素- 5 -13℃; 1 -15 ñ </td><td> CDN的同位素</td><td> M - 2307 </td><td>批号＃L340P9 </td></tr><tr><td> MTBSTFA </td><td> Thermo Scientific的</td><td> 48920 </td><td></td></tr><tr><td>吡啶</td><td> Sigma - Aldrich公司</td><td> 270970 </td><td></td></tr><tr><td> 5973E的GC / MSD </td><td>安捷伦科技公司</td><td> G7021A </td><td>零件编号为5975E的GC / MS </td></tr><tr><td> 3，Ethoxymethacrolein </td><td> Sigma - Aldrich公司</td><td> 232548 </td><td></td></tr><tr><td>氢氧化钠</td><td> Sigma - Aldrich公司</td><td> S5881 </td><td></td></tr><tr><td>二氯甲烷</td><td> Sigma - Aldrich公司</td><td> 270997 </td><td></td></tr><tr><td>苯</td><td> Sigma - Aldrich公司</td><td> 401765 </td><td></td></tr><tr><td>异丙醚</td><td> Sigma - Aldrich公司</td><td> 38270 </td><td></td></tr><tr><td> BHT </td><td> Sigma - Aldrich公司</td><td> B1378 </td><td></td></tr><tr><td>乙醇</td><td> Sigma - Aldrich公司</td><td> 459844 </td><td></td></tr><tr><td>苯</td><td> Sigma - Aldrich公司</td><td> P26252 </td><td></td></tr></tbody></table>

biochemical measurement of neonatal hypoxia

新生儿缺氧缺血的特点是一个组织的血流灌注不足或全身的氧气缺乏。这种情况被认为是造成/加剧据可查的新生儿疾病包括神经受损 1-3 。三磷酸腺苷生产下降的发生是由于缺乏氧化磷酸化的。为了弥补这种能量被剥夺的状态，含有高能磷酸债券分子退化 2 。这导致腺苷水平的提高，这是随后降解为肌苷，次黄嘌呤，黄嘌呤，最后以尿酸。黄嘌呤氧化还原酶在这个降解过程中的最后两个步骤进行。这种酶黄嘌呤脱氢酶的形式存在，常氧条件下，但转换下再灌注缺氧情况下，4，5黄嘌呤氧化酶（ XO）。与黄嘌呤脱氢酶，XO笔记本电脑生成过氧化氢 ​​作为一种嘌呤降解4，6的副产品。这与其他缺氧时产生的活性氧（ROS），结合过氧化氢 ​​的氧化尿酸形成尿囊素和脂质膜反应生成丙二醛（MDA）7-9。大多数哺乳动物，人类豁免，具有酶的尿酸， 尿酸转换尿囊素。然而，在人类中，尿囊素只能形成尿酸ROS介导的氧化。正因为如此，尿囊素被认为是人体氧化应激的标记，但在哺乳动物尿酸 。 我们描述了采用高压液相色谱（HPLC）和气相色谱质谱（GCMS）的方法来衡量新生儿缺氧缺血的生化标志物，人体血液是大多数测试使用。 动物血也可以使用，同时也认识到尿酸生成尿囊素的潜力。嘌呤代谢物与缺氧早在1963年和次黄嘌呤，黄嘌呤的可靠性，并为新生儿缺氧的生化指标的尿酸是由10-13几个调查验证。用于嘌呤类化合物的定量的高效液相色谱法快速，可靠，重现性好。 GC / MS方法用于量化尿囊素，一个相对较新的标志物的氧化应激，适应格鲁伯 等 7 。这种方法避免了一定的文物和需要的样品量低。描述其他地方所采用的方法合成MMDA 14，15。改编自Paroni 等的GC / MS MDA基于量化。 Cighetti 等 16，17。黄嘌呤氧化酶活性测定高效液相色谱法，通过量化蝶呤转换isoxanthopterin 18 。这种方法被证明是足够的敏感和重复性。

Watch this Scientific Journal Video about Biochemical Measurement of Neonatal Hypoxia at JoVE.com

Biochemical Measurement of Neonatal Hypoxia

描述的方法来衡量新生儿缺氧缺血的生化标志物。该方法利用高压液相色谱（HPLC）和气相色谱质谱（GC / MS）。

新生儿缺氧的生化测量

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