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，并根据血浆可用总量的嘌呤卷。 </li></ol> 2。准备内部标准，2 - 氨基嘌呤（2 - AP），嘌呤和XO分析<ol><li>称0.01351克2 - AP的，将它添加到过的水与盐酸2-5滴酸化8毫升。如果不溶于2 - AP，您需要添加更多的酸。调整最终体积至10ml。 </li><li>使用紫外可见分光光度计，以确定你的股票2 - AP解决方案的真实浓度。 （315的λmax，ε4000）。 </li><li>一旦股票的浓度2 - AP的解决方案是确定的，计算含有1 × 10 -7摩尔2 - AP内部嘌呤测量标准和1 × 10 -8 MOL 2 - AP内部的XO测量标准所需的卷。 </li> 公式1： <img src="/files/ftp_upload/2948/2948eq1.jpg" alt="公式1" /> 公式（2）： <img src="/files/ftp_upload/2948/2948eq2.jpg" alt="公式（2）" /><li>分装成不同的离心管的计算量，并在Speed​​Vac蒸发至干。 </li></ol> 3。高效液相色谱法测量嘌呤<ol><li>传输血浆（200μL）一个单片机YM - 10离心式过滤装置和离心机在4 ° C时为1.5小时的14000克。 </li><li>取出滤液转移到微量离心管中，含有1 × 10 -7摩尔2 - AP 。请务必记录滤液体积的离心管中，含有2 - AP。涡10-20秒的样品。 </li><li>用HPLC分析的样本。三50μL注射用于每个样品。样品注入到一个Supelcosil LC - 18 - S，15厘米× 4.6毫米，5μm色谱柱配备一个Supelguard LC - 18 - S列后卫。应使用表1中所描述梯度条件获得足够的峰的分离度：一个水溶剂，溶剂B甲醇，溶剂的C - 50MM酸铵缓冲液，pH值5.5。 </li></ol><table border="1"><tbody><tr><td>&nbsp;</td><td> 时间 </td><td> 流量（毫升/分钟） </td><td> ％A </td><td> ％B级 </td><td> ％C </td></tr><tr><td> 1 </td><td></td><td> 1.00 </td><td> 0.0 </td><td> 0.0 </td><td> 100.0 </td></tr><tr><td> 2 </td><td> 16.00 </td><td> 1.00 </td><td> 0.0 </td><td> 0.0 </td><td> 100.0 </td></tr><tr><td> 3 </td><td> 17.00 </td><td> 1.00 </td><td> 100.0 </td><td> 0.0 </td><td> 0.0 </td></tr><tr><td> 4 </td><td> 22.00 </td><td> 1.00 </td><td> 100.0 </td><td> 0.0 </td><td> 0.0 </td></tr><tr><td> 5 </td><td> 27.00 </td><td> 1.00 </td><td> 0.0 </td><td> 100.0 </td><td> 0.0 </td></tr><tr><td> 6 </td><td> 32.00 </td><td> 1.00 </td><td> 0.0 </td><td> 100.0 </td><td> 0.0 </td></tr><tr><td> 7 </td><td> 33.00 </td><td> 1.00 </td><td> 100.0 </td><td> 0.0 </td><td> 0.0 </td></tr><tr><td> 8 </td><td> 38.00 </td><td> 1.00 </td><td> 100.0 </td><td> 0.0 </td><td> 0.0 </td></tr><tr><td> 9 </td><td> 39.00 </td><td> 1.00 </td><td> 0.0 </td><td> 0.0 </td><td> 100.0 </td></tr><tr><td> 10 </td><td> 45.00 </td><td> 1.00 </td><td> 0.0 </td><td> 0.0 </td><td> 100.0 </td></tr></tbody></table>为嘌呤类化合物的高效液相色谱法测量见表1。溶剂的变化。 <ol start="4"><li>确定样品中的嘌呤浓度。次黄嘌呤，黄嘌呤，尿酸量化取得的保留和表2，确定2 - AP的峰面积的波长峰面积。面积比，次黄嘌呤，黄嘌呤和尿酸确定2 - AP和转换比率，使用标准曲线μmolar浓度。 </li></ol><table border="1"><tbody><tr><td>&nbsp;</td><td> 保留时间* </td><td> 观察λ 最大 * </td><td> 报道λ 最大 19 </td><td> 报道ε 最大 19 </td></tr><tr><td> 尿酸酸</td><td> 〜3.5敏</td><td> 288 </td><td> 283 [2] </td><td> 11500 [2] </td></tr><tr><td> 次黄嘌呤 </td><td> 〜7.0敏</td><td> 248 </td><td> 248 [1] </td><td> 10800 [1] </td></tr><tr><td> 黄嘌呤 </td><td> 〜9.5分钟</td><td> 267 </td><td> 267 [2] </td><td> 10200 [2] </td></tr><tr><td> 2 -氨基嘌呤 </td><td> 〜12.5分钟</td><td> 305 </td><td> 314 [2] </td><td> 4000 [2] </td></tr></tbody></table> 表2典型的保留时间和λ最大嘌呤和内部标准。 *对高效液相色谱法测定使用的为1ml/min的流速等度50MM酸铵缓冲液（pH值5.5）。 pH值是在[]。 <ol start="5"><li>分析所有样本一式三份，但只包括以供日后分析值的变异系数小于10％。 </li></ol> 4。尿囊素的GC / MS测定<ol><li>添加50μL10μMDL -尿囊素- 5 - 13℃; 1 - 15 N（内部标准）50μL血浆预留尿囊素检测过程中样品的采集和处理。 </li><li>加入100μL乙腈这种等离子体的解决方案。 </li><li>旋涡混合10-20秒，然后离心10分钟在4 ° C的20,000克。 </li><li>取出上清液，放置在一个小瓶的GC / MS和干下N 2。 </li><li>干燥后，加入50μL衍生剂N -叔丁基二甲基- N - methyltrifluoroacetamide（MTBSTFA）吡啶（1:1体积/体积），第小瓶，并培育他们在50 ° 2小时彗星衍生产量坚持量化为398.0和400.0米/ Z峰;尿囊素和DL -尿囊素- 5 - 13 C 1 - 15 N，分别为20。 </li><li>分析样品，安捷伦6890N气相色谱和质谱5973配备了自动采样器。执行化合物的分离在Agilent 122 - 5532G的毛细管柱（25.7米长度，0.25毫米内部直径）。使用氦气作为载气，在流速为1.5毫升/分钟。注入分割模式（分流比20:1，分流29.4毫升/分，总流量33.8毫升/分）的衍生产品（1μL）。初始柱温设置为100 ° C和保持在该温度下2分钟，然后增加至180 ° C时，税率为10 ° C /分钟。保持温度为4分钟，再增加至260 ° C率在20 ° C /分钟。保持这个温度，直到运行结束。每个样品后，2注射正己烷清洗之列。 </li><li>量化尿囊素使用DL -尿囊素- 5 - 13 C选择离子监测模式，同时监测了398.0米/ Z离子的尿囊素和400.0 m / z为离子; 1 - 15 N.尿囊素/（重尿囊素）的离子丰度比转换尿囊素微摩尔浓度，用事先准备好的标准曲线。 </li><li>所有样品进行了分析，一式三份，但低于10％的变异系数的值只有在进一步分析使用。 </li></ol> 5。 MDA的分析准备的内部标准，甲基丙二醛（MMDA）， <ol><li>在100毫升的圆底烧瓶中，加入523μl3 ethoxymethacrolein1477μl7M氢氧化钠。添加一个stirbar。 </li><li>烧瓶置于水浴中，在45℃，搅拌，直到反应已经完成。这应该需要大约140分钟。在10分钟的间隔中删除的液体中加入10μl等份定期监测反应的进展。稀释收集每个样本由10 5 50 mM磷酸钾缓冲液（pH值7），测量的UV - Vis吸收的因素。一旦达到大约0.658的反应完全在275nm的吸光度。随着反应的进行，该解决方案将变成黄色，然后橙色。 </li><li>允许的反应进度为一个额外的15分钟。 </li><li>圆底烧瓶中加入蒸馏水去离子水5毫升和解决方案转移至分液漏斗。使用3毫升的水，洗成漏斗的烧瓶中的内容。添加额外的水2毫升漏斗。 </li><li>用5毫升的二氯甲烷提取解决方案的3倍。每次提取后丢弃的有机层。 </li><li>第三提取后，水层转移到圆底烧瓶中，并rotovap干燥。 </li><li>悬浮在3毫升乙醇和产品转移到一个预先称重的15毫升锥形管。额外2毫升乙醇冲洗烧瓶中，并添加冲洗锥形管。 </li><li>加入5ml苯和孵化管在温水中，直到产品溶解，然后立即置于冰上10分钟管。离心5分钟，15000克，去除上清。 </li><li>加5ml乙醇和5毫升异丙醚的降雨雪itant。溶解的沉淀孵化管，并定期在温水中轻轻倾斜管。一旦被溶解析出，放置在冰管10分钟。执行此用乙醇和异丙醇醚重结晶2次以上。有些产品可能会形成不溶性的白色块。 </li><li>最后再结晶步骤后删除尽可能多的可能和干燥造成的产品在Speed​​vac上清。权衡与合成产品的锥形管。 </li><li>加5ml水，管，涡街，并过滤掉任何剩余的固体。 </li><li>使用紫外可见分光光度计，以确定最终MMDA的浓度。 MMDA在50毫米磷酸钾缓冲液（pH值7）的λmax为274 nm的消光系数为29900 M -1厘米-1 14。 </li></ol> 6。 MDA的气相色谱/质谱测量<ol><li>准备0.5MM二丁基羟基甲苯（BTH）在乙醇溶液中加入0.11克BTH9毫升乙醇和调整最终体积至10ml。然后执行通过这种浓缩液990μl乙醇的加入中加入10μl稀释。 </li><li>准备在一个黑暗的离心管中加入苯肼4.92μl水995μl的50MM苯肼的解决方案。涡的解决方案。 </li><li>添加5μL10μMMMDA100μL血浆MDA的检测过程中样品的采集和处理拨出。 </li><li>然后添加加入10μl0.5MM BTH此外200μL1M柠檬酸钠在 pH 4 MMDA /等离子混合物pH值是正确的样品衍生的关键。 较高或较低的pH值将导致歪斜MDA水平。 </li><li>加入蒸馏水去离子水到终体积为480μl稀释的最终混合物。 </li><li>加入20μL50MM苯肼，封盖的小瓶和解决方案，在25 ° C孵育30分钟轨道摇床的Derivati​​ze解决方案。 </li><li>添加正己烷，1分钟，10分钟3000转离心旋涡1毫升。 </li><li>有机层转移到小瓶的GC / MS和集中由N 2流的解决方案，以100μL。 </li><li>使用自动采样器上的GC / MS分析的样品。执行复合分离在Agilent 122 - 5532G毛细管柱（25.7米长，内部直径0.25毫米）。使用氦气作为载气，在0.6-0.8毫升/分钟的流速。注入分割模式（分流比20:1，分流23.0毫升/分，总流量27.1毫升/分）的衍生产品（2μL）。设置初始柱温为110 ° C和在该温度下保持1分钟前提高到250 ° C率在40 ° C /分钟。保持这个温度，直到运行结束。每个样品后，2注射正己烷清洗之列。 </li><li>量化MDA的使用选择离子监测模式使用的144.00米/ MDA的Z和158.00米/ MMDA ž。转换的MDA / MMDA离子丰度比使用微摩尔浓度的标准曲线。 </li><li>分析样品一式三份，并只使用值的变异系数小于10％。 </li></ol> 7。高效液相色谱法测定黄嘌呤氧化酶<ol><li>检测黄嘌呤氧化酶的功能是依赖于能够灵敏地测量一个合适的基板（蝶呤），以及其产品（isoxanthopterin）的水平。培养底物为4小时0分（对照组）或血浆，并确定向产品转化的孵化依赖基板。 </li><li>加入240μl0.2 M Tris - HCl缓冲液（pH 9.0）和4.63μl7.083x10 -3 M蝶呤，以每管（0和4h）和preincubate在37℃下5分钟。 </li><li>启动每管加入60μl血浆酶反应。 </li><li>立即加入300μLHCLO 4 4％的控制管（0分），但允许其他混合物孵育淬火前4小时反应300μL4％HCLO 4 。 </li><li>此外HCLO 4后，大力摇晃管，然后在4 ° C时，15000克离心15分钟。 </li><li>取出上清液500μL和消除5M K 2 CO 3加入20μL。大力摇晃管，然后在4 ° C时，15000克离心15分钟。 </li><li>加入350μl的瓦解解决微量离心管中，含有1 × 10 -8 MOL 2 - AP 。 </li><li>配备扫描荧光检测样品的HPLC分析。三50μL注射用于每个样品。样品将被注入到一个Wacosil 5C - 18 - 200 250毫米× 6.0毫米，5微米配备一个Supelguard LC - 18 - S列后卫列。应采用以下的等度条件下获得足够的峰的分离和鉴定：95％20MM KPO 4缓冲液（pH值2.2）和5％的甲醇在流速1ml/min。设置在345 nm处的荧光检测的激发波长和发射波长长度为410 nm。 </li><li>确定的pterine和isoxanthopterine 2 - AP获得荧光检测光谱峰面积的比率。频谱中的第一个高峰，〜5分钟，相当于2 - AP。第二和最大峰值将pterine。 Isoxanthopterine高峰洗脱去年。 </li><li>获取的isoxanthopterine/2-AP峰面积比0和孵育4 h时间点之间的差异。使用此值来计算从标准曲线的XO活动。 </li><li>分析样本一式三份，但使用唯一值的变异系数小于10％。 </li></ol> 8。代表性的成果： 嘌呤类化合物的高效液相色谱定量的一个例子如图 1a所示。具体的保留时间和发射波长的次黄嘌呤，黄嘌呤，尿酸许可证的嘌呤类化合物的同时的定量（见表2）。检测是正常运行时，该化合物将有足够的分离和峰形尖锐和单峰。这些峰值，然后将其转换成浓度范围如表3所示，通过使用标准曲线。因为这个实验中的样品处理是最小的，只有基于样本可能出现的问题将红细胞裂解。如果红血细胞裂解样品离心前，等离子将采取橙色/红色，不能用于评估缺氧缺血。在测量嘌呤时可能出现的的其他问题涉及的高效液相色谱系统和列（ 图 1B ）。如果有气泡在高效液相色谱系统的保留时间将转向和高效液相色谱法的压力将大幅波动。如果后卫墨盒需要改变，压力将会增加，峰将扩大，并成为双向或三模态。 <table border="1"><tbody><tr><td></td><td>次黄嘌呤（微米） </td><td>黄嘌呤（微米） </td><td>尿酸（微米） </td><td>黄嘌呤氧化酶（（nmol产品）/分钟） </td><td>丙二醛（微米） </td><td>尿囊素（微米） </td></tr><tr><td>长期常氧</td><td> 1.13-19.3（64） </td><td> 0.02-3.69（61） </td><td> 107.20-726.12（63） </td><td> 2.47x10 -6 - 3.92x10 -3（20） </td><td> 0.44-3.76（53） </td><td>不适用</td></tr><tr><td>期呼吸窘迫</td><td> 1.78-12.59（27） </td><td> 0.07-11.8（24） </td><td> 225.40-653.32（27） </td><td> 0 - 1.03x10 -5（8） </td><td> 0.82-2.73（24） </td><td>不适用</td></tr><tr><td>长期缺氧</td><td> 0.38-31.80（13） </td><td> 0.11-2.88（13） </td><td> 235.65-1348.13（13） </td><td> 1.20x10 -5 -236.44（9） </td><td> 0.95-2.15（7） </td><td>不适用</td></tr><tr><td>早产儿常氧</td><td> 1.54-4.39（9） </td><td> 0.03-1.77（9） </td><td> 178.92-593.49（9） </td><td> 2.46x10 -5 - 5.44x10 -4（2） </td><td> 0.95-2.74（8） </td><td> 2.30-5.26（67） </td></tr><tr><td>早产儿呼吸困境</td><td> 3.04-8.04（2） </td><td>不适用</td><td> 327.56-365.11（2） </td><td>不适用</td><td> 2.40-3.46（3） </td><td>不适用</td></tr></tbody></table>最小 - 最大（N） 表3。嘌呤，黄嘌呤氧化酶，丙二醛和尿囊素的代表范围。 尿囊素的GC / MS定量的一个例子是如图 2所示。由于derivitized尿囊素和derivitized重尿囊素的质量是已知的，选择离子模式可以被用来确定这些化合物的质谱。如果检测是正确的，两峰将在相同保留时间的观察。一个相应的尿囊素（398.00 M / Z）和其他重尿囊素（400.00 M / Z）。这些峰值，然后将其转换成浓度范围如表3所示，通过使用标准曲线。如果实验运行不正确，样本不正确derivitized，峰值可能不存在或可能不定量的代表性。再次，如果红细胞裂解等离子不能被用来评价新生儿缺氧缺血氧化应激。 MDA的量化结果尿囊素那些与观察到的异常是在不同的保留时间，两峰的相似。应〜3.5分钟的保留时间，144.00 m / z为MDA和高峰〜4分钟的保留时间，一个158.00米/ Z峰值为MMDA观察（ 图3）。这些峰值，然后将其转换成浓度范围如表3所示，通过使用标准曲线。如果实验运行不正确，或样本是不derivitized正确，无峰144.00米/ z和158.00米/ Z选择时，可观察应该指出，如果有超过丸口服喂食或静脉注射脂质管理的血浆脂质，血浆将采取一种乳白色的外观，并不能用来评估新生儿缺氧缺血氧化应激。 高效液相色谱法为基础的定量黄嘌呤氧化酶的功能的一个例子是如图4所示。如果检测是正常运行，三个峰，应观察与荧光检测器，一个为蝶呤isoxanthopterin之一，2 - AP之一。这些峰值，然后将其转换成浓度范围如表3所示，通过使用标准曲线。还应该有一个峰值对应isoxanthopterin和2 - AP从PDA检测器所产生的频谱。如果缺少酶的活性，对应isoxanthopterin高峰将不可见。由于这种检测措施酶的功能，反复冻融和样品解冻，可能会改变这一点。 <img src="/files/ftp_upload/2948/2948fig1a.jpg" alt="图1a"/> <img src="/files/ftp_upload/2948/2948fig1b.jpg" alt="图1b"/> 图1。高效液相色谱法谱嘌呤类化合物的鉴定。 A）。代表性的结果，如果检测正常运行。 B）。代表性的结果，如果有一个问题，用高效液相色谱仪，列，或保护盒。 <img src="/files/ftp_upload/2948/2948fig2.jpg" alt="图2"/> 图2。 GC / MS谱尿囊素的量化 。离子398.00峰的m / z对应尿囊素。离子400.00米/ Z的峰值对应于沉重尿囊素。 <img src="/files/ftp_upload/2948/2948fig3.jpg" alt="图3"/> 图3。 GC / MS的量化MDA的频谱。离子144.00米/ Z峰值对应到MDA。离子158.00米/ Z峰值对应MMDA。 <img src="/files/ftp_upload/2948/2948fig4.jpg" alt="图4"/> 图4。高效液相色谱法光谱测量的XO活动。）代表0分钟的孵育时间和孵化时间为4小时b）代表结果结果。请注意，较高的孵化时间为4小时isoxanthopterine高峰（〜17分钟）。在荧光光谱中，2 - AP流出第一〜5min和pterine流出〜10分钟的未来。

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没有利益冲突的声明。

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

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

biochemical-measurement-of-neonatal-hypoxia

Research

JoVE Journal

Medicine

14.0K 次观看.  Loma Linda University. 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）。

视频: Biochemical Measurement of Neonatal Hypoxia

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

新生儿窒息了猪模型

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

科研

医学

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

小鼠模型的腔内MCAO脑梗塞评价甲酚紫染色

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

婴幼儿的脑代谢和血流动力学的非侵入式光学测量

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. Collecting site-specific biochemical markers is important to understand the relationship between levels in serum and surgical wound, determine any associations between mediator release, pain, analgesic use and other outcomes of interest, and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry. This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. We have measured wound exudate and serum mediators at the same time intervals as patient's pain scores and analgesics consumption for up to 48 hours post-cesarean delivery. Using this methodology we have been able to detect various biochemical mediators including nerve growth factor (NGF), prostaglandin E2 (PG-E2) substance P, IL-1&#x03B2;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&#x03B3;, G-CSF, GM-CSF, MCP-1 and MIP-1&#x03B2;. Studies applying this human surgical wound bioassay have found no correlations between wound and serum cytokine concentrations or their time-release profile (J Pain. 2008; 9(7):650-7).1 We also documented the utility of the technique to identify drug-mediated changes in wound cytokine content (Anesth Analg 2010; 111:1452-9).2

This article provides a detailed and visual description of a methodology for collecting and measuring biochemical inflammatory and nociceptive mediators at the surgical wound site following cesarean delivery. This human bioassay has been used to determine correlations between wound and serum cytokine concentrations and drug-mediated changes in wound cytokines, chemokines and neuropetides.

手术伤口伤口渗出液生化调解员在收集和测量

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

心率在小鼠中通过无创M型超声心动图测量子宫内

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生儿缺氧缺血性脑病的小猪模型

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

同时PET / MRI成像在小鼠脑缺氧缺血

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

基于短暂性脑缺氧缺血性血栓性中风模型

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

一个模型来模拟人类临床相关性缺氧

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...