Name: measurement of tissue non-heme iron content using a bathophenanthroline-based colorimetric assay
Uploaded: 2022-01-31T14:45:00
Description: Watch this Scientific Journal Video about Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay at JoVE.com

这项工作由国家基金通过FCT-Fundação para a Ciência e a Tecnologia，I.P.资助，在UIDB/04293/2020项目下。

C57BL / 6小鼠是商业购买的，C57BL / 6背景上的铁调素空（Hamp1−/−）小鼠8 是Sophie Vaulont（法国科钦研究所）的善意礼物。动物被安置在i3S动物设施中，在特定的无病原体条件下，在温度和光线受控的环境中，可以自由获取标准的啮齿动物食物和水。欧洲鲈鱼（Dicentrarchus labrax）是从一个商业养鱼场购买的，饲养在ICBAS动物设施中，在温度和光线受控的环境中，...

<ol>
	<li>Muckenthaler, M. U., Rivella, S., Hentze, M. W., Galy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+red+carpet+for+iron+metabolism.">A red carpet for iron metabolism.</a> Cell. 168, 344-361 (2017).</li><li>Pagani, A., Nai, A., Silvestri, L., Camaschella, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepcidin+and+anemia:+A+tight+relationship.">Hepcidin and anemia: A tight relationship.</a> Frontiers in Physiology. 10, 1294 (2019).</li><li>Weiss, G., Ganz, T., Goodnough, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anemia+of+inflammation.">Anemia of inflammation.</a> Blood. 133 (1), 40-50 (2019).</li><li>Altamura, S., 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=Regulation+of+iron+homeostasis:+Lessons+from+mouse+models.">Regulation of iron homeostasis: Lessons from mouse models.</a> Molecular Aspects of Medicine. 75, 100872 (2020).</li><li>Torrance, J. D., Bothwell, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+measuring+storage+iron+concentrations+in+formalinised+liver+samples.">A simple technique for measuring storage iron concentrations in formalinised liver samples.</a> South African Journal of Medical Sciences. 33 (1), 9-11 (1968).</li><li>Torrence, J. D., Bothwell, T. H., Cook, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+iron+stores.">Tissue iron stores.</a> Methods in Haematology. , 104-109 (1980).</li><li>Rebouche, C. J., Wilcox, C. L., Widness, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microanalysis+of+non-heme+iron+in+animal+tissues.">Microanalysis of non-heme iron in animal tissues.</a> Journal of Biochemical and Biophysical Methods. 58 (3), 239-251 (2004).</li><li>Lesbordes-Brion, J. C., 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=Targeted+disruption+of+the+hepcidin+1+gene+results+in+severe+hemochromatosis.">Targeted disruption of the hepcidin 1 gene results in severe hemochromatosis.</a> Blood. 108, 1402-1405 (2006).</li><li>Jumbo-Lucioni, P., 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=Systems+genetics+analysis+of+body+weight+and+energy+metabolism+traits+in+Drosophila+melanogaster.">Systems genetics analysis of body weight and energy metabolism traits in Drosophila melanogaster.</a> BMC Genomics. 11, 297 (2010).</li><li>Mandilaras, K., Pathmanathan, T., Missirlis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+Absorption+in+Drosophila+melanogaster.">Iron Absorption in Drosophila melanogaster.</a> Nutrients. 5, 1622-1647 (2013).</li><li>Grundy, M. A., Gorman, N., Sinclair, P. R., Chorney, M. J., Gerhard, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+non-heme+iron+assay+for+animal+tissues.">High-throughput non-heme iron assay for animal tissues.</a> Journal of Biochemical and Biophysical Methods. 59, 195-200 (2004).</li><li>Adrian, W. J., Stevens, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wet+versus+dry+weights+for+heavy+metal+toxicity+determinations+in+duck+liver.">Wet versus dry weights for heavy metal toxicity determinations in duck liver.</a> Journal of Wildlife Diseases. 15, 125-126 (1979).</li></ol>

提供了测量动物组织中非血红素铁含量的方案，使用最初由Torrance和Bothwell描述的基于菲罗啉的比色测定法的适应性5，6。该方法的关键步骤是组织样品干燥;蛋白质变性并通过酸水解释放无机铁;在还原剂硫代乙醇酸存在下将铁（Fe3 +）还原为亚铁状态（Fe2 +），并将其与菲咯啉试剂反应（染色原反应）;所得黑色离子/菲咯啉粉红色复合物?...

铁是一种必需的微量营养素，是参与关键生物过程（如氧气运输、能量产生或DNA合成）的蛋白质功能所必需的。重要的是，铁过量和缺铁都对人体健康非常有害，组织铁水平受到精细调节。饮食中铁吸收异常、缺铁饮食、反复输血和慢性炎症是影响全球数十亿人的铁相关疾病的常见原因1，2，3。
铁超负荷或缺乏的实验动物模型有助于提高我们对铁稳态的全身和细胞调节机制的了解4。尽管在过去二十年中取得了重大进展，但许多关键方面仍然难以捉摸。在未来几年，准确测量动物组织中的总铁水平仍将是推进铁生物学领域研究的关键一步。
大多数实验室使用原子吸收光谱（AAS），电感耦合血浆质谱（ICP-MS）或基于非血红素铁与菲螺母试剂反应的比色测定来定量组织铁。后者基于Torrance和Bothwell在50多年前描述的原始方法5，6。虽然随后开发了该方法的变体，采用亚铁嗪作为菲咯啉的替代品7，但后者仍然是文献中引用最广泛的显色试剂。
选择的方法通常取决于可用的专业知识和基础结构。虽然AAS和ICP-MS更敏感，但比色测定仍然被广泛使用，因为它具有以下重要优点：i）它排除了来自红细胞中含有的血红蛋白的血红素铁的贡献;ii）它不需要复杂的分析技能或昂贵的设备;iii）原始的基于比色皿的测定可以适应微孔板形式，从而实现更高的样品通量。这项工作中提出的比色方法通常用于量化从啮齿动物到鱼和果蝇的各种铁超载或缺铁实验动物模型中组织非血红素铁水平的变化。在这里，提供了一种测量动物组织中非血红素铁含量的方案，使用简单，成熟的比色测定法，大多数实验室应该发现易于实施。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well UV transparent plate</td>
			<td>Sarstedt</td>
			<td>82.1581.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Kern</td>
			<td>ABJ 220-4M</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous sodium acetate</td>
			<td>Merck</td>
			<td>106268</td>
			<td></td>
		</tr>
		<tr>
			<td>Bathophenanthroline sulfonate (4,7-Diphenyl-1,10-phenantroline dissulfonic acid)</td>
			<td>Sigma-Aldrich</td>
			<td>B1375</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice (Mus musculus)</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonyl iron powder, &#8805;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>44890</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable cuvettes in polymethyl methacrylate (PMMA)</td>
			<td>VWR</td>
			<td>634-0678P</td>
			<td></td>
		</tr>
		<tr>
			<td>Double distilled, sterile water</td>
			<td>B. Braun</td>
			<td>0082479E</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microplate reader</td>
			<td>BioTek Instruments</td>
			<td>FLx800</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 37%</td>
			<td>Sigma-Aldrich</td>
			<td>258148</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave digestion oven and white teflon cups</td>
			<td>CEM</td>
			<td>MDS-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric acid</td>
			<td>Fisher Scientific</td>
			<td>15687290</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Binder</td>
			<td>ED115</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent chow</td>
			<td>Harlan Laboratories</td>
			<td>2014S</td>
			<td>Teklad Global 14% Protein Rodent Maintenance Diet containing 175 mg/kg iron</td>
		</tr>
		<tr>
			<td>Sea bass (Dicentrarchus labrax)</td>
			<td>Sonrionansa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sea bass feed</td>
			<td>Skretting</td>
			<td>L-2 Alterna 1P</td>
			<td></td>
		</tr>
		<tr>
			<td>Single beam UV-Vis spectrophotometer</td>
			<td>Shimadzu</td>
			<td>UV mini 1240</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioglycolic acid</td>
			<td>Merck</td>
			<td>100700</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid</td>
			<td>Merck</td>
			<td>100807</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of tissue non-heme iron content using a bathophenanthroline-based colorimetric assay

铁是一种必需的微量营养素。铁超负荷和缺乏都对人类非常有害，组织铁水平受到精细调节。铁超负荷或缺乏的实验动物模型的使用有助于提高铁稳态的全身和细胞调节机制的知识。动物组织中总铁水平的测量通常使用原子吸收光谱或基于非血红素铁与菲罗啉试剂反应的比色测定进行。多年来，比色测定法一直用于测量各种动物组织中的非血红素铁含量。与原子吸收光谱不同，它排除了来自红细胞中含有的血红蛋白的血红素铁的贡献。此外，它不需要复杂的分析技能或昂贵的设备，因此可以在大多数实验室中轻松实施。最后，比色法可以基于比色皿，也可以适应微孔板形式，从而实现更高的样品通量。本工作提供了一个完善的方案，适用于检测铁超载或缺铁的各种实验动物模型中组织铁水平的改变。

比色皿与 96 孔微孔板比较 通过与最初由Torrance和Botthwell5，6描述的菲咯啉试剂反应测量组织非血红素铁依赖于使用分光光度计进行吸光度读数。因此，染色原反应中使用的体积与常规分光光度计比色皿的大小相容。本工作描述了一种方法适应性，其中染色原反应直接在96孔微孔板中制备，用于在酶标仪中测量吸光度，需要更小的试剂体积?...

Watch this Scientific Journal Video about Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay at JoVE.com

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

在这里，提供了一种测量动物组织中非血红素铁含量的方案，使用一种简单，成熟的比色法，可以在大多数实验室中轻松实施。

使用基于菲咯啉的比色法测量组织非血红素铁含量

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...

在这里，我们提供了一个用于测量动物组织中非血红素含量的方案。使用简单且完善的比色测定法，可在大多数实验室中轻松实施。基于菲罗啉的比色法不需要复杂的技能或昂贵的设备，并且可以适应微孔板形式，以获得更高的样品通量和成本效益。该协议适用于检测铁过载或铁效率的各种实验动物模型中组织铁水平的改变。首先用手术刀刀片切割重达10至100克的组织样本。在分析天平中，在一小块胶片上准确称量。使用塑料镊子将组织片放入24孔板中。让它在65摄氏度的标准培养箱中干燥48小时。或者，使用粘镊子将涉水组织片放入无铁特氟龙杯中，然后在实验室微波消化烤箱中干燥。将每块干燥的组织放在分析天平内的一小块模板上，并准确称量。将每块干燥的组织转移到1.5毫升的微量离心管中。将点燃的酸混合物加入管中并关闭。以相同的方式准备酸坯，除了此处省略了组织。将管子在65摄氏度下孵育20小时以消化组织。冷却至室温后，使用装有塑料吸头的微量移液器将500微升透明酸提取物转移到新的1.5毫升微量离心管中。将染色原反应直接制备到平底96孔中，透明未经处理的聚苯乙烯微孔板中。室温孵育15分钟。在离离子水参考下，以535纳米的波长测量读板器中的样品吸光度。此处显示了通过比色皿和96孔基于微孔板的比色测定法测定非血红素铁。这里显示了来自6个月大雄性黑，6只小鼠（包括肝脏，脾脏，心脏，肺和骨髓）的55个组织样本中基于比色皿和基于微孔板的非血红素铁水平之间的相关性。两种方法之间的高度相关性表明，基于微孔板的方法是原始比色皿方法的有效替代方案。最后，用基于微孔板的方法对来自相同组织的样品进行酸性消解后，用盐酸和三氯乙酸或单独盐酸的混合物对非血红素铁水平进行定量。高相关性表明，三氯乙酸可以从酸消解中释放出来。代表性图像显示了雄性黑六野生型小鼠和雄性铁调素敲除小鼠在黑六遗传背景上用基于菲罗林的比色法测量的非血红素铁水平。铁调素的破坏导致血色病，如铁沉积表型，伴有严重的肝、胰腺和心脏铁蓄积以及脾铁耗竭。这里显示了实验性铁调节后欧洲鲈鱼幼年雌性的肝脏非血红素铁水平。在铁治疗动物中，肝铁水平显着增加。而贫血导致肝铁储存轻度减少。代表性图像显示了用基于菲罗啉的比色法测量的一个月大果蝇的非血红素铁水平。结果显示了每组20只苍蝇中的非血红素铁含量或每只苍蝇的估计铁含量。考虑到每只苍蝇的平均重量为0.64毫克。在尝试此过程时，请记住彻底清洁所有玻璃磨损，并且由于存在污染风险，不要让金属实验室材料与任何试剂或溶液接触。

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