This work was funded by National Funds through FCT-Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia, I.P., under the project UIDB/04293/2020.

C57BL/6 mice were commercially purchased and hepcidin-null (Hamp1&#8722;/&#8722;) mice on a C57BL/6 background8 were a kind gift from Sophie Vaulont (Institut Cochin, France). Animals were housed at the i3S animal facility under specific pathogen-free conditions, in a temperature- and light-controlled environment, with free access to standard rodent chow and water. European sea bass (Dicentrarchus labrax) were purchased from a commercial fish farm and housed at the ICB...

<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>

A protocol for the measurement of the non-heme iron content in animal tissues is provided, using an adaptation of the bathophenanthroline-based colorimetric assay originally described by Torrance and Bothwell5,6. The critical steps of the method are tissue sample drying; protein denaturation and release of inorganic iron by acid hydrolysis; reduction of ferric (Fe3+) iron to the ferrous state (Fe2+) in the presence of the reducing agent thio...

Iron is an essential micronutrient, required for the function of proteins involved in crucial biological processes such as oxygen transport, energy production, or DNA synthesis. Importantly, both iron excess and iron deficiency are highly detrimental to human health, and tissue iron levels are finely regulated. Abnormal dietary iron absorption, iron-deficient diets, repeated blood transfusions, and chronic inflammation are common causes of iron-associated disorders that affect billions of people worldwide1,2,3.
Experimental animal models of iron overload or deficiency have been instrumental to advance our knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis4. Despite the substantial progress made during the last two decades, many key aspects remain elusive. In the coming years, the accurate measurement of total iron levels in animal tissues will remain a critical step to advance research in the iron biology field.
Most laboratories quantify tissue iron with either atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS), or a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. The latter is based on the original method described by Torrance and Bothwell over 50 years ago5,6. While a variation of this method was subsequently developed employing ferrozine as an alternative to bathophenanthroline7, the latter remains the most widely cited chromogenic reagent in the literature.
The method of choice often depends on the available expertise and infrastructure. While AAS and ICP-MS are more sensitive, the colorimetric assay remains widely used because it presents the following important advantages: i) it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells; ii) it does not require sophisticated analytical skills or highly expensive equipment; and iii) the original cuvette-based assay can be adapted to a microplate format, allowing higher sample throughput. The colorimetric approach presented in this work is routinely used to quantify alterations in tissue non-heme iron levels in a variety of experimental animal models of iron overload or iron deficiency, from rodents to fish and fruit fly. Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established, colorimetric assay that most laboratories should find easy to implement.

<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

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Cuvette versus 96-well microplate comparison 
The measurement of tissue non-heme iron by reaction with a bathophenanthroline reagent originally described by Torrance and Bothwell5,6 relies on the use of a spectrophotometer for absorbance reading. Hence, the volumes employed in the chromogen reaction are compatible with the size of a regular spectrophotometer cuvette. The present work describes a method adaptation in which the chromogen react...

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

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

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

Here we have provided a protocol for measuring the non-Heme contents in animal tissues. Using a simple well-established colorimetric assay that can be implemented easily in most laboratories. The Bathophenanthroline-Based Colorimetric Assay does not require sophisticated skills or highly expensive equipment and can be adapted to microplate format for higher sample throughput and cost effectiveness.This protocol is suited for detecting alterations in tissue iron levels in a variety of experimental animal models of iron overloads or iron efficiency. Begin by cutting a tissue sample weighing 10 to 100 grams with a scalpel blade. Weigh it accurately in an analytical balance over a small piece of parafilm.Use plastic tweezers to place the piece of tissue in a 24 well plate. Let it dry on a standard incubator at 65 degree celsius for 48 hours. Alternatively place the wade piece of tissue using stick tweezers in an iron free Teflon cup and dry it in the laboratory microwave digestion oven.Place each dried piece of tissue over a small piece of paraform inside an analytical balance and weigh it accurately. Transfer each dried piece of tissue into a 1.5 milliliter micro centrifuge tube. Add a lit of the acid mixture to the tube and close it.Prepare an acid blank in the same way except that the tissue is omitted here. Incubate the tubes at 65 degrees celsius for 20 hours to digest the tissue. After cooling to room temperature use a micro pipette fitted with plastic tips to transfer 500 microliters of the clear acid extract into a new 1.5 milliliter micro centrifuge tube.Prepare chromogen reactions directly into the flat bottom 96-well, clear untreated polystyrene micro plates. Incubated room temperature for 15 minutes. Measure sample absorbance in a plate reader at a wavelength of 535 nano meters against a deionized water reference.Determination of non-heme iron by the cuvette and 96-well microplate based colorimetric assays are shown here. The correlation between cuvette based and micro plate based non-heme iron levels in 55 tissue samples from six month old male black, six mice, including liver, spleen, heart, lungs and bone marrow are shown here. The high correlation between the two methodologies indicates that the micro plate based method is a valid alternative to the original cuvette based method.Finally, non-heme iron levels were quantified with the micro plate based method after acidic digestion of sample derived from the same tissues with either a mixture of hydrochloric acid and trichloroacetic acid or hydrochloric acid alone. The high correlation shows that trichloroacetic acid can be emitted from the acid digestion. The representative image shows the non-heme iron levels in the liver, spleen, heart and pancreas of male black six wild type mice and male hepcidin knockout mice on black six genetic background measured with Bathophenanthroline-Based Colorimetric Assay.The disruption of hepcidin leads to a hemochromatosis like iron deposition phenotype with severe hepatic, pancreatic and cardiac iron accumulation and splenic iron depletion. Hepatic non-heme iron levels in juvenile female European sea bass following experimental iron modulation are shown here. Hepatic iron levels increased massively in iron treated animals.Whereas anemia caused a mild reduction in the hepatic iron stores. The representative images show the non-heme iron levels in one month old Drosophila measured with a Bathophenanthroline-Based Colorimetric Assay. Results show the non-heme iron content in each pool of 20 flies or the estimated iron content of each fly.Considering an average weight of 0.64 milligrams per fly. While attempting this procedure remember to thoroughly clean all the glass wear and do not allow the metallic laboratory materials to come into contact with any reagent or solution due to the risk of contamination.

measurement-tissue-non-heme-iron-content-using-bathophenanthroline

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JoVE Journal

Medicine

4.0K visualizações.  Universidade do Porto. Aqui fornecemos um protocolo para medir o conteúdo não-Heme em tecidos animais. Usando um simples estonteante assimétrico colorido que pode ser implementado facilmente na maioria dos laboratórios. O ensaio Colorimétrico Baseado em Bathophenanthroline não requer habilidades sofisticadas ou equipamentos altamente caros e pode ser adaptado ao formato de microplape para maior rendimento amostral e custo-benefício.Este protocolo é adequado para detectar alterações nos níveis de f...