Name: evaluation of oxidative stress in biological samples using the thiobarbituric acid reactive substances assay
Uploaded: 2020-05-12T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay at JoVE.com

这里报告的研究得到了美国国立卫生研究院国家癌症研究所的部分支持。R33 CA217702和最大化学生发展倡议（IMSD）计划。内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。

人类血清标本是在IRB批准下根据《赫尔辛基宣言》中表达的原则从同意的志愿者那里获得的。在转移到分析实验室之前，对标本进行编码和去鉴定。
1. 样品制备
<ol><li>七氢呋喃二酮细胞裂解物
	<ol>
<li>将每个烧瓶接种约10×10 6 个HepG2细胞放入16个T75烧瓶中，加入14mL EMEM培养基，补充10%胎牛血清（FBS），并培养细胞2天。</li>
		<li>制备 RIPA 缓冲液:在 50 mL 管中，加入 1.5 mL 5 M NaCl、2.5 mL 1 M Tris-HCl （pH = 7）、500 μL NP-40 试剂，然后用 DI 水将最终体积增至 50 mL。</li>
		<li>制备裂解缓冲液:将20mL RIPA缓冲液等分到50mL管中，并加入200μL100x蛋白酶抑制剂溶液以抑制蛋白质和脂质降解。储存在4°C。 注意:裂解缓冲液与TBARS试剂兼容，不会干扰532nm处的吸光度。如果计划使用不同的裂解缓冲液或向裂解缓冲液中添加其他成分，则需要进行初步验证研究以验证裂解缓冲液组分是否与TBARS测定兼容。</li>
		<li>取出含有10%FBS的培养基，用5 mL冷无菌1x PBS洗涤细胞2x。</li>
		<li>向含有细胞的T75烧瓶中加入1mL裂解缓冲液，并在室温（RT）下孵育10分钟，并持续旋转以确保缓冲液分布良好。</li>
		<li>将裂解物收集到适当标记的2 mL卡扣盖聚丙烯管中，并在冰上孵育10分钟。</li>
		<li>在室温下以5，000×g旋转裂解物10分钟以收集细胞碎片，并将上清液吸入单个15 mL管中。</li>
		<li>在50°C和3 mbar下使用Speed Vac将细胞裂解物上清液浓缩四倍，并将94μL的等分试样放入2 mL卡扣帽聚丙烯管中。将样品储存在-80°C，直到用于体外氧化和/或TBARS测定。 注意:为避免浓缩细胞裂解物上清液，也可以使用3mL1x胰蛋白酶分离细胞，用6mL培养基中和，并用5mL冷PBS洗涤2x。然后可以在250μL裂解缓冲液中重构细胞沉淀，然后可以执行步骤1.1.6和1.1.7。</li>
		<li>在乙酸中制备35mM CuCl2 储备溶液（pH = 4）。<ol>
<li>准备乙酸溶液（pH = 4）:将1μL冰醋酸稀释在100mL的DI水中（pH值应约为4，但用pH计确认）。加入更多的水或乙酸，将pH值调节到4。</li>
			<li>取出约0.1936克氯化铜，溶于10毫升乙酸溶液（pH = 4）中，制成144 mM CuCl2 储备液。从该溶液中等分490μL，并加入1，510μL乙酸（pH = 4）以制成35mM CuCl2 溶液。</li>
		</ol>
</li>
		<li>从35 mM CuCl2 储备溶液中等分6μL，并将其加入含有94μL细胞裂解物的6个样品中，使最终CuCl2 浓度约为2mM。将6μL不含CuCl2 的乙酸溶液（pH = 4）加入含有94μL细胞裂解物的六个样品中作为对照。细胞裂解物的最终体积应为100μL，这是用于TBARS测定的体积。 注意:在乙酸（pH = 4）中制造35 mM CuCl2 储备溶液对于防止氢氧化铜沉淀是必要的。</li>
		<li>将样品在37°C的烘箱中孵育24小时，并对含有100μL最终体积的每个样品进行TBARS测定。</li>
		<li>在不同的日子里重复步骤1.1.9和1.1.11 2x，以检查HepG2细胞裂解物的TBARS测定的再现性。</li>
	</ol></li>
	<li>低密度脂蛋白 注意:通常，预纯化的低密度脂蛋白（LDL）含有一定量的EDTA。这里使用的LDL样品含有0.01%的EDTA。EDTA可以抑制体外Cu（II）介导的LDL氧化。因此，在实验或分析之前，可能需要从LDL样品中除去EDTA。步骤 1.2.1–1.2.5 描述了此过程。 注意:氢氧化钠具有腐蚀性，会刺激皮肤和眼睛。使用适当的个人防护装备。<ol>
<li>将24μL从5.51mg / mL低密度脂蛋白储备液（使用BSA作为标准通过改良的Lowry方法测定蛋白质浓度）等分到适当标记的1 mL卡扣帽聚丙烯管中。根据需要制作尽可能多的等分试样，并储存在4°C，直到用于氧化和/或TBARS测定。</li>
		<li>用NaOH微珠在0.15 M NaCl中制备10mM HEPES缓冲液，调节至pH = 7:将4.39gNaCl溶解在0.49L水中，然后加入1.19gHEPES。用搅拌棒充分溶解。加入氢氧化钠珠，直到pH值为7。用水稀释至0.5升。将缓冲液储存在4°C，并在3个月内使用。</li>
		<li>将476μL的10mM HEPES缓冲液加入0.15M NaCl（pH = 7）到等分的LDL样品中，使最终体积达到500μL。将稀释的LDL样品加入具有100K分子量截止值的0.5mL离心离心过滤装置中。</li>
		<li>在室温下以14，000×g离心样品10分钟，留下约30μL的最终截留物体积。将样品在480μL的10mM HEPES缓冲液中在0.15 M NaCl（pH = 7）中重构，并在室温下以14，000×g再次离心10分钟。</li>
		<li>将过滤装置倒置到新的2mL卡扣盖聚丙烯管中，并以1000×g离心2分钟以收集LDL样品（最终体积= 约30μL）。</li>
		<li>将样品等分到适当标记的1 mL管中，并向每个样品中加入20μL水，以达到50μL的最终体积。</li>
		<li>在乙酸中制备200μM CuCl2 储备溶液（pH = 4）<ol>
<li>制备乙酸溶液（pH = 4）:参见步骤1.1.9.1。</li>
			<li>准备144mM CuCl2 储备溶液（见步骤1.1.9.2），然后从144mM CuCl2 储备液中等分5.5μL，溶解在最终体积为4mL乙酸（pH = 4）中以制成200μM溶液。</li>
		</ol>
</li>
		<li>从200μM CuCl2储备溶液中等分2.7μL，并将其加入到含有50μLL的六个样品中，以达到〜10μM的最终CuCl2浓度。将不含CuCl2的乙酸溶液（pH = 4）中的2.7μL加入到含有50μLLL的六个样品中用于对照。</li>
		<li>将LDL样品在37°C的烘箱中孵育2小时。 2小时后，用10mM HEPES缓冲液在0.15M NaCl（pH = 7）中将每个样品的最终体积降至100μL。立即进行 TBARS 检测。</li>
		<li>在两个不同的日期重复步骤1.2.3–1.2.9 2x，以测试TBARS测定的再现性。</li>
	</ol></li>
	<li>人血清
	<ol>
<li>从人血清样品中，将94μL等分试样放入2mL卡扣盖聚丙烯管中，并将样品储存在-80°C。</li>
		<li>在乙酸中制备35mM CuCl2 储备溶液（pH = 4）:参见步骤1.1.9。</li>
		<li>从CuCl2 储备溶液中等分6μL，并将其加入含有94μL人血清的6个样品中，使最终CuCl2 浓度约为2mM。将6μL不含CuCl2 的乙酸溶液（pH = 4）加入含有94μL人血清的六个样品中作为对照使用。</li>
		<li>将人血清样品在37°C的烘箱中孵育24小时，并用TBARS测定法测定氧化水平（第4节）。</li>
		<li>在两天内重复步骤1.3.2–1.3.4 2x，以确定TBARS测定的再现性。</li>
	</ol></li>
</ol>2. 试剂制备
注意:硫代巴比妥酸会引起皮肤和眼睛刺激，吸入或皮肤吸收可能有害。醋酸如果吸入会损害内脏器官。在通风橱中准备所有酸溶液。
<ol><li>制备8.1%（w / v）十二烷基硫酸钠（SDS）溶液<ol>
<li>取出 32.4 g SDS 的重量，溶于烧杯中的 350 mL 去离子水。使用磁性搅拌棒轻轻溶解SDS并避免产生气泡。用去离子水将最终体积调至400 mL，并将SDS溶液储存在室温下。 注意:在这里，准备了过量的8.1%SDS溶液;然而，对于96个样品，只需要约20 mL的8.1%SDS溶液。根据要分析的样品数量准备此溶液。</li>
	</ol>
</li>
	<li>制备3.5M乙酸钠缓冲液（pH = 4）<ol>
<li>在烧杯中将 100 mL 冰醋酸稀释到 350 mL 去离子水中。使用磁性搅拌棒轻轻溶解。</li>
		<li>在水中使用氢氧化钠珠制备6.5M NaOH溶液。将 13 g NaOH 微珠溶解在 40 mL 的 DI 水中，并用 DI 水使最终体积达到 50 mL。</li>
		<li>在与搅拌棒混合时，缓慢地向乙酸溶液中加入约46mL的6.5M NaOH溶液（这应该将pH提高到4，但在使用pH计测量时缓慢加入NaOH溶液来确认）。</li>
		<li>用去离子水将最终体积降至500 mL，并将乙酸钠缓冲液储存在室温下。</li>
	</ol>
</li>
	<li>制备0.8%硫代巴比妥酸水溶液（调节至pH = 4） 注意:在此步骤中，硫代巴比妥酸的制备针对大体积进行了优化，因为将要分析大量样品（108个样品，不包括标准品）。根据计划用于分析的样品数量准备此解决方案。<ol>
<li>使用氢氧化钠珠和水制备5M氢氧化钠溶液:将4g氢氧化钠珠溶于最终体积的20mL水中。存放在塑料容器中。该溶液应为每批新鲜制备。</li>
		<li>重量4克硫代巴比妥酸，加入450毫升去离子水。使用磁性搅拌棒轻轻溶解。 注:该溶液最终将达到500 mL的总体积。</li>
		<li>当用搅拌棒溶解硫代巴比妥酸时，以100μL的增量（缓慢和滴滴方式）加入约3mL的5M NaOH溶液。加入NaOH溶液后，硫代巴比妥酸颗粒将开始溶解。</li>
		<li>如果硫代巴比妥酸颗粒仍未完全溶解，则以100μL的增量加入更多的5M NaOH溶液，直到所有硫代巴比妥酸颗粒完全溶解。对于这种特定体积的溶液，共加入4mL的5M NaOH溶液以完全溶解硫代巴比妥酸颗粒。 注意:在此浓度下，硫代巴比妥酸不会完全溶解，除非pH值接近4。</li>
		<li>在所有硫代巴比妥酸完全溶解后停止添加NaOH。避免超过pH值4。可以通过从混合的硫代巴比妥酸溶液中取1μL并将其置于pH纸上来验证最终的pH值。</li>
		<li>用去离子水将最终体积降至500 mL，并在室温下储存0.8%硫代巴比妥酸水溶液。</li>
	</ol>
</li>
</ol>3. 丙二醛双（缩二甲醇）标准样品制备
注意:丙二醛（MDA）不稳定，无法在市场上买到。然而，市面上有不同的化学形式的MDA，例如MDA四丁基铵盐，MDA双（二甲缩醛）和MDA双（缩二乙醇）。在这三种化学形式中，这里使用MDA双（二甲缩醛），因为大多数研究使用相同的标准21，22。如果选择使用其他两种化学形式的MDA，应事先验证其适用性。
<ol><li>通过将92μL纯MDA双（二甲基缩醛）稀释在1L二甲基苯丙水中，制备550μM MDA双（二甲基缩醛）储备溶液。使用磁性搅拌棒将溶液彻底混合10分钟。将溶液储存在4°C并在1个月内使用。</li>
	<li>通过将550μM MDA双（二甲缩醛）储备液中的726μL稀释到1274μL的DI水中，制备200μM MDA双（二甲缩醛）。每次进行TBARS测定时，应新鲜制备这种200μM MDA双（缩二甲醇）溶液。</li>
	<li>标准曲线制备:取8个2 mL卡扣帽聚丙烯管，并用字母A至H标记，从200μM储备液中加入MDA双（二甲缩醛），并按 表1所述在水中稀释。</li>
	<li>取8个玻璃管（13 mm x 100 mm）并标记它们A-H，然后将100μL标准品加入相应的管中。对空白标准品（样品A）执行六次重复，以计算TBARS测定的检测限。 注意:该协议可以在此处暂停不超过1小时。</li>
</ol>4. TBARS检测
注意:一旦开始TBARS测定，就应该在不停止的情况下完成。
<ol><li>根据需要取尽可能多的玻璃管，以分析样品的数量，并用样品名称标记它们。然后，将100μL每个制备的样品（如上所述）加入每个玻璃管中。</li>
	<li>向每个样品和标准品中加入200μL8.1%SDS，并以圆周运动轻轻旋转玻璃管以混合样品。</li>
	<li>向每个样品和标准品中加入1.5 mL的3.5 M乙酸钠缓冲液（pH = 4）。</li>
	<li>向每个样品和标准品中加入1.5mL的0.8%硫代巴比妥酸水溶液（pH = 4）。</li>
	<li>通过加入700μL的DI水，将每个样品的最终体积和标准品的最终体积降至4 mL。</li>
	<li>紧紧盖住每个玻璃管，并在设置为95°C的加热块中孵育1小时。用铝箔盖住玻璃管，以防止管顶部结露。</li>
	<li>从加热块中取出玻璃管，在冰上孵育30分钟。</li>
	<li>在4°C下以1500×g离心样品和标准品10分钟。 离心后，将含有样品和标准品的玻璃管保持在室温。 注意:将样品保存在冰上或4°C会导致整个样品或标准品沉淀。</li>
	<li>离心后立即，从每个管中等分150μL上清液，并放入96孔板的单独孔中。</li>
	<li>使用移液器吸头从每个孔中除去任何气泡。 注意:气泡的存在会产生不一致的吸光度读数，从而导致高度的测定不精确性。</li>
	<li>读取532nm处的吸光度。从所有其他吸光度读数中减去空白样品的平均吸光度读数。</li>
	<li>通过绘制532nm处的空白减去吸光度读数与每种标准的已知浓度来创建标准曲线。使用线性回归拟合数据点。通过使用从标准曲线获得的线性回归线方程计算未知样品浓度。</li>
</ol>

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作者没有竞争性经济利益或其他利益冲突需要披露。

尽管 TBARS 检测方法存在局限性1、3、4、7、8、9、10、12、13、14、15、19 且不适合在实验室?...

脂质过氧化是自由基（如活性氧和活性氮）攻击脂质中的碳 - 碳双键的过程，该过程涉及从碳中抽取氢并插入氧分子。该过程导致复杂产物的混合物，包括脂质过氧自由基和氢过氧化物作为主要产物，以及丙二醛（MDA）和4-羟基壬烯醛作为主要的二次产物1。
MDA因其与硫代巴比妥酸（TBA）的易反应而被广泛用于生物医学研究中，作为脂质过氧化的标志物。该反应导致MDA-TBA2的形成，MDA-TBA2是一种共轭物，在532nm处的可见光谱中吸收并产生红粉红色2。除MDA外，来自脂质过氧化的其他分子也可以与TBA反应并在532nm处吸收光，从而有助于测量的整体吸收信号。同样，MDA可以与大多数其他主要类别的生物分子反应，可能限制其与TBA3，4反应的可及性。因此，这种传统的测定方法仅被认为可以测量"硫代巴比妥酸反应物质"或TBARS5。
当正确应用和解释时，TBARS测定通常被认为是生物样品中氧化应激总体水平的良好指标6。不幸的是，正如Khoubnasabjafari和其他人所记录的那样，TBARS测定的进行和解释方式往往有助于得出可疑的结论3，4，7，8，9，10，11。造成这种情况的原因主要在于与样品相关的预分析变量，以及缺乏检测坚固性，从而禁止在检测方案中发生看似微小的变化，而不会对测定结果进行实质性更改1，7，12，13。
与生物标本处理和储存相关的预分析变量（例如，暂时保持在-20°C的血浆）14，15 可对TBARS测定结果产生重大影响16，17;如此之多，以至于TBARS测定结果不应在不同实验室之间进行比较，除非有明确的实验室间分析验证数据保证。此建议类似于蛋白质印迹的常用和解释方式。条带密度的比较对于印迹和实验室内研究是有效的，但比较实验室之间的条带密度通常被认为是一种无效的做法。
一些研究人员认为，通过TBARS测定法测量的MDA根本不符合可接受的生物标志物所需的分析或临床标准3，9，10，18，19。事实上，如果该测定法不是在50多年前开发的，它可能不会获得今天的广泛使用和默认接受。尽管还有其他具有更高分析灵敏度、特异性和坚固性的测定方法用于测定氧化应激，但基于532 nm吸光度的 TBARS 测定仍然是迄今为止最常用的测定脂质过氧化的测定方法之一20，从而评估氧化应激。
TBARS测定只能作为昂贵的试剂盒（超过400美元）找到，其中说明书没有提供有关所用试剂的大多数浓度的详细信息。此外，所提供的试剂只能用于一个实验，因为每个试剂盒只能做一个比色标准曲线。对于打算在不同时间点确定几个样品中的氧化水平的研究人员来说，这可能是个问题，因为相同的标准曲线不能多次使用。因此，需要购买多个试剂盒进行多个实验。目前，除非购买昂贵的试剂盒，否则没有有关如何进行TBARS测定的详细方案。过去的一些研究人员模糊地描述了如何进行TBARS测定21，22，但文献中既没有关于如何在没有昂贵试剂盒的情况下进行TBARS测定的完全详细的实验方案或全面的视频。
在这里，我们报告了一种详细的，经过分析验证的用于目的的方法，该方法涉及如何以简单，可重复且廉价的方式进行TBARS测定。用Cu（II）离子处理后人血清，HepG2裂解物和低密度脂蛋白的脂质过氧化变化被证明为TBARS测定的说明性应用。结果表明，这种TBARS测定在日常基础上是一致且可重复的。

<table><tbody><tr><td>1x Sterile PBS pH 7.4 1 L</td><td>VWR, PA</td><td>101642--262</td><td>cell lysis reagent</td></tr><tr><td>50 mL self-standing centrifuge tube</td><td>Corning, NY</td><td>CLS430897</td><td>General material</td></tr><tr><td>96 well plate, Non-Treated, clear, with lid, Non-sterile</td><td>Thermo Fisher Scientific, MA</td><td>280895</td><td>To measure absorbance</td></tr><tr><td>Amicon Ultra-0.5 100 kD centrifugal spin filter device</td><td>Fisher Scientific, NH</td><td>UFC510024</td><td>LDL purification</td></tr><tr><td>Caps for glass tubes</td><td>Thermo Fisher Scientific, MA</td><td>14-930-15D</td><td>for TBARS assay</td></tr><tr><td>Copper II Chloride</td><td>SIGMA, MO</td><td>222011-250G</td><td>to induce oxidation</td></tr><tr><td>Culture tubes, Disposable, with Screw-Cap Finish, Borosilicate Glass (13 x 100 mm)</td><td>VWR, PA</td><td>53283-800</td><td>for TBARS assay</td></tr><tr><td>Eagle&#039;s Minimum Essential Medium (EMEM)</td><td>ATCC, VA</td><td>HB-8065</td><td>HepG2 cell media</td></tr><tr><td>Eppendorf Safe-Lock Tubes, 1.5 mL</td><td>eppendorf, NY</td><td>22363204</td><td>General material</td></tr><tr><td>Eppendorf Safe-Lock Tubes, 2.0 mL</td><td>Genesee Sceitific, CA</td><td>22363352</td><td>General material</td></tr><tr><td>Fetal Bovine Serum US Source</td><td>Omega Scientific, CA</td><td>FB-11</td><td>for cell culture</td></tr><tr><td>Glacial Acetic Acid</td><td>SIGMA, MO</td><td>27225-1L-R</td><td>TBARS Reagent</td></tr><tr><td>Halt Protease Inhibitor Cocktail (100x)</td><td>Thermo Scientific, MA</td><td>87786</td><td>cell lysis reagent</td></tr><tr><td>HEPES</td><td>SIGMA, MO</td><td>H3375-250G</td><td>LDL solvent</td></tr><tr><td>HepG2 Cells</td><td>ATCC, VA</td><td>HB-8065</td><td>Biological matrix prototype</td></tr><tr><td>Hydrocloric acid (HCl)</td><td>Fisher Scientific, NH</td><td>A144-212</td><td>cell lysis reagent</td></tr><tr><td>Legend Micro 17 Centrifuge</td><td>Thermo Scientific, MA</td><td>75002431</td><td>General material</td></tr><tr><td>Low Density Lipoprotein, Human Plasma</td><td>Athens Research &amp;amp; Technology, GA</td><td>12-16-120412</td><td>Biological matrix prototype</td></tr><tr><td>Magnetic Stir Bars, Octagon 6-Assortment</td><td>VWR, PA</td><td>58948-025</td><td>General material</td></tr><tr><td>Malondialdehyde bis (dimethyl acetal)</td><td>SIGMA, MO</td><td>8207560250</td><td>TBARS Standard</td></tr><tr><td>Multiskan Go Microplate Spectrophotometer</td><td>Fisher Scientific, NH</td><td>51119200</td><td>To measure absorbance</td></tr><tr><td>NP-40</td><td>EMD Millipore Corp, MA</td><td>492016-100ML</td><td>cell lysis reagent</td></tr><tr><td>Sodium Chloride</td><td>SIGMA, MO</td><td>S7653-1KG</td><td>cell lysis reagent</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>SIGMA, MO</td><td>436143-100G</td><td>TBARS Reagent</td></tr><tr><td>Sodium hydroxide</td><td>SIGMA, MO</td><td>367176-2.5KG</td><td>TBARS Reagent</td></tr><tr><td>SpeedVac Concentrator</td><td>Thermo Scientific, MA</td><td>SC250EXP</td><td>For concentrating cell lysates</td></tr><tr><td>T-75 Flask, Tissue Culture Treated, 250 mL, w/filter cap</td><td>USA Scientific, FL</td><td>658175</td><td>cell culture</td></tr><tr><td>Thiobarbituric Acid</td><td>SIGMA, MO</td><td>T5500-100G</td><td>TBARS Reagent</td></tr><tr><td>TRIS base</td><td>Fluka, GA</td><td>93362</td><td>cell lysis reagent</td></tr><tr><td>Trypsin (1x)</td><td>VWR, PA</td><td>16777-166</td><td>To detach HepG2 cells</td></tr></tbody></table>

evaluation of oxidative stress in biological samples using the thiobarbituric acid reactive substances assay

尽管硫代巴比妥酸反应物质（TBARS）测定的分析特异性和坚固性有限，但已被广泛用作生物体液中脂质过氧化的通用指标。它通常被认为是生物样品中氧化应激水平的良好指标，前提是样品已得到妥善处理和储存。该测定涉及脂质过氧化产物（主要是丙二醛（MDA））与硫代巴比妥酸（TBA）的反应，这导致形成称为TBARS的MDA-TBA2加合物。TBARS产生红粉红色，可以在532nm处进行分光光度法测量。TBARS测定在酸性条件（pH = 4）和95°C下进行。 纯MDA是不稳定的，但这些条件允许MDA双（二甲缩醛）释放MDA，MDA在该方法中用作分析标准。TBARS测定是一种简单的方法，可以在大约2小时内完成。此处详细介绍了测定试剂的制备。注重预算的研究人员可以低成本将这些试剂用于多个实验，而不是购买昂贵的TBARS检测试剂盒，该试剂盒仅允许构建单个标准曲线（因此只能用于一个实验）。该TBARS测定的适用性在人血清，低密度脂蛋白和细胞裂解物中显示。该测定结果一致且可重复，检测限可达到 1.1 μM。提供了分光光度法TBARS测定的使用和解释的建议。

在酸性条件（pH = 4）和95°C下，丙二醛（MDA）双（二甲缩醛）产生MDA23。MDA和密切相关的化学同源物与两个硫代巴比妥酸分子（TBA）反应，产生称为硫代巴比妥酸反应物质（TBARS）的化合物，其呈红粉红色，在532nm处具有吸光度λmax （图1， 图2）。以MDA bis（缩二甲醇）为标准，生成标准曲线（图3， ?...

Watch this Scientific Journal Video about Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay at JoVE.com

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

硫代巴比妥酸反应物质测定的目的是通过使用可见光波长分光光度法在532nm处测量脂质过氧化产物（主要是丙二醛）的产生来评估生物样品中的氧化应激。这里描述的方法可应用于人血清、细胞裂解物和低密度脂蛋白。

使用硫代巴比妥酸反应物质测定法评估生物样品中的氧化应激

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic ...

evaluation-oxidative-stress-biological-samples-using-thiobarbituric

Research

JoVE Journal

Chemistry

27.9K 次观看.  Arizona State University. 硫代巴比妥酸反应物质测定的目的是通过使用可见光波长分光光度法在532nm处测量脂质过氧化产物（主要是丙二醛）的产生来评估生物样品中的氧化应激。这里描述的方法可应用于人血清、细胞裂解物和低密度脂蛋白。

视频: Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

科研

癌症研究

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

生物化学

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

<meta charset="utf8"></head><body>评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

环境科学

Hellgrammite as a Non-Conventional Biomonitor for Surface Water and Environmental Health Assessment

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of occurrence, and extended period of immaturity, which can last up to one year. Hellgrammites are clearly known to exhibit sensitivity to pollution and habitat structure impacts. Given these unique features, using Corydalus texanus larvae is highly suitable as reliable biomonitoring agents to assess the ecological integrity of aquatic ecosystems. This protocol aims to provide the necessary tools for C. texanus assessment and demonstrate their efficacy through a case study. Research findings have practical implications, indicating that C. texanus larvae exhibit early-warning responses to mining pollution, bioaccumulating high amounts of heavy metals such as Zn, Fe, and Al. The presence or absence of C. texanus populations may serve as a helpful indicator for identifying potential issues related to ecosystem health. The unconventional approach has shown early warnings of pollution in mining-impacted sites, highlighting the need for timely action to protect the environment. Given their unique traits, the use of C. texanus larvae is highly suggested as a reliable non-conventional bioindicator.

This protocol describes the use of biomarkers for the early detection of deleterious impacts in aquatic ecosystems. The biomarkers are closely related to sentinel traits, and their changes aid in detecting early-warning damages.

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of ...

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. 
The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.

树脂辅助捕获与同量异位串联质量标签标记相结合，用于蛋白质硫醇氧化的多重定量

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed ...

GSH/GSSG 测量 – 用于抗氧化剂研究的改进 OPA 检测

Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30&#181;M (R2=0.9932&#177;0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.

作者聚焦：创新巯基定量和生物标志物检测用于氧化应激研究 

Quantification of both oxidized and reduced forms of glutathione (GSSG and GSH, respectively) has been achieved through the use of Ortho-phthalaldehyde (OPA). OPA becomes highly fluorescent once conjugated to GSH but is unable to conjugate GSSG until reduced. Here, we describe a multiparametric assay to quantify both using protein quantification for normalization.

在体外使用 邻 苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

使用硫代巴比妥酸反应物质测定法评估生物样品中的氧化应激

摘要

探索更多视频

此视频中的章节

用基因编码荧光传感器评估毒理学氧化应激的成像方法

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

Hellgrammite as a Non-Conventional Biomonitor for Surface Water and Environmental Health Assessment

树脂辅助捕获与同量异位串联质量标签标记相结合，用于蛋白质硫醇氧化的多重定量

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

利用同位素标记的质谱剖析巯基氧化还原蛋白质

使用硫代巴比妥酸反应物质测定法评估生物样品中的氧化应激

摘要

探索更多视频

此视频中的章节

用基因编码荧光传感器评估毒理学氧化应激的成像方法

通过2'，7'-二氯二氢氟辛二乙酸染色检测粘附细胞中的总活性氧物种

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

Hellgrammite as a Non-Conventional Biomonitor for Surface Water and Environmental Health Assessment

树脂辅助捕获与同量异位串联质量标签标记相结合，用于蛋白质硫醇氧化的多重定量

在体外使用 邻 苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用 邻 苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用 邻 苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

利用同位素标记的质谱剖析巯基氧化还原蛋白质

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽

在体外使用邻苯二甲醛在培养的哺乳动物细胞中快速定量氧化和还原形式的谷胱甘肽