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 ICBAS animal facility, in a temperature- and light-controlled environment, and fed daily ad libitum with standard sea bass feed. All procedures involving vertebrate animals were approved by the i3S Animal Ethics Committee and the national authority, Dire&#231;&#227;o-Geral de Alimenta&#231;&#227;o e Veterin&#225;ria (DGAV). Information about commercial reagents, equipment, and animals is listed in the Table of Materials.
1. Solution preparation
NOTE: Handle and prepare all reagents and solutions with iron-free glassware or disposable plasticware. Do not allow metallic laboratory materials (e.g., stainless steel spatulas) to come in contact with any reagent or solution, due to the risk of iron contamination. Make sure any reusable glassware is iron-free. Wash the materials with appropriate laboratory detergent for 30-60 min, rinse with deionized water, soak overnight in a 37% nitric acid solution diluted 1:3 with deionized water, rinse again with deionized water, and allow to dry.
<ol>
	<li>Acid mixture: Add 10 g of trichloroacetic acid to 82.2 mL of 37% hydrochloric acid in a glass bottle, thoroughly dissolve, and adjust the final volume to 100 mL with deionized water. Shake before use. Alternatively, use only 37% hydrochloric acid for tissue digestion. 
	NOTE: The solution is stable for at least 2 months when stored in dark brown glass reagent bottles. 
	CAUTION: Hydrochloric acid and trichloroacetic acid are corrosive, and concentrated forms release toxic acidic vapors. Wear protective garments, chemical-resistant gloves, and chemical splash goggles at all times when handling acids. Avoid breathing them in and always handle acids while under a fume hood.</li>
	<li>Saturated sodium acetate: Add 228 g of anhydrous sodium acetate to 400 mL of deionized water in a glass bottle and agitate overnight at room temperature. Let the solution rest and precipitate for a day. If no precipitation occurs, continue adding small amounts of sodium acetate. Store the solution in a glass bottle.</li>
	<li>Chromogen Reagent: To prepare 1 mL of chromogen reagent, add 1 mg of 4,7-diphenyl-1,10-phenanthroline disulfonic acid disodium salt to 500 &#181;L of deionized water and 10 &#181;L of concentrated (100%) thioglycolic acid, and thoroughly dissolve. Make up the final volume to 1 mL with deionized water. 
	NOTE: Prepare as much chromogen reagent as needed. The solution is stable for 1 month when protected from light.</li>
	<li>Working Chromogen Reagent (WCR): Add 1 volume of the Chromogen Reagent to 5 volumes of saturated sodium acetate and 5 volumes of deionized water. 
	NOTE: This solution should be prepared freshly on the day of use.</li>
	<li>Stock Iron Standard Solution: To prepare a 20 mM stock iron solution, place 111.5 mg of carbonyl iron powder in a 250 mL volumetric flask containing 5,480 &#181;L of 37% hydrochloric acid. Leave to dissolve overnight at room temperature (or incubate in a boiling water bath). Then, make up the solution to a final volume of 100 mL with deionized water. 
	NOTE: The standard solution can be kept indefinitely when stored in a tightly sealed vessel.</li>
	<li>Working Iron Standard Solution (WISS): Add 13.5 &#181;L of 37% hydrochloric acid to 500 &#181;L of deionized water. Add 10 &#181;L of the Stock Iron Standard Solution and make up the final volume to 1 mL with deionized water (11.169 &#181;g of Fe/mL, 200 &#181;M; measured by AAS). 
	&#8203;NOTE: The working solution should be prepared freshly on the day of use.</li>
</ol>
2. Sample drying
<ol>
	<li>Cut a sample of tissue weighing 10-100 mg with a scalpel blade. Weigh it accurately in an analytical/precision balance over a small piece of parafilm (Fresh Weight).</li>
	<li>Using plastic tweezers, place the piece of tissue in a 24-well plate (unlidded to allow for water evaporation) and let it dry on a standard incubator at 65 &#176;C for 48 h.</li>
	<li>Alternatively, use a laboratory microwave digestion oven for drying tissue samples. Using plastic tweezers, place the weighed piece of tissue in an iron-free Teflon cup and dry it in the microwave. Set the operating parameters according to the instrument&#39;s instructions manual. 
	NOTE: As a reference, operating parameters for drying liver samples using the specific digestion oven (see Table of Materials) are shown in Table 1.</li>
	<li>Using plastic tweezers, place each dried piece of tissue over a small piece of parafilm inside an analytical/precision balance and weigh it accurately (Dry Weight).</li>
</ol>
3. Sample acidic digestion
<ol>
	<li>Using plastic tweezers, transfer each dried piece of tissue into a 1.5 mL microcentrifuge tube.</li>
	<li>Add 1 mL of the acid mixture and close the microcentrifuge tube. Prepare an acid blank in the same way, except that the tissue is omitted. 
	CAUTION: The acid mixture is corrosive and releases toxic vapors. Wear protective garments, chemical-resistant gloves, and chemical splash goggles when handling the acid mixture. Avoid breathing it in and always handle it while under a fume hood.</li>
	<li>Digest the tissues by incubating the microcentrifuge tubes in an incubator at 65 &#176;C for 20 h.</li>
	<li>After cooling to room temperature, transfer 500 &#181;L of the clear (yellow) acid extract (supernatant) into a new 1.5 mL microcentrifuge tube using a micropipette fitted with plastic tips. If it is not possible to obtain a clear supernatant, perform a short centrifugation spin. 
	CAUTION: The supernatant is highly acidic. Wear protective garments, chemical-resistant gloves, and chemical splash goggles when handling the supernatants. Always handle them while under a fume hood. 
	&#8203;NOTE: At this point, the acid extracts can be immediately used for the colorimetric assay or frozen at -20 &#176;C for later use. Completely thaw frozen samples to room temperature and vortex them prior to use.</li>
</ol>
4. Color development
<ol>
	<li>Prepare chromogen reactions as indicated in Table 2 in 1.5 mL microcentrifuge tubes or, for higher throughput, directly into the flat bottom, 96-well, clear, untreated polystyrene microplates. Prepare all reactions (acid blank, standard, and sample) at least in duplicate.</li>
	<li>Incubate at room temperature for 15 min.</li>
</ol>
5. Absorbance reading
<ol>
	<li>Measure sample absorbance in a spectrophotometer or plate reader at a wavelength of 535 nm against a deionized water reference. Plates can be read unlidded or lidded. In the lidded case, remove any condensation formed due to the release of acid vapors from the lid just prior to the measurement to avoid possible interference with the absorbance reading. 
	&#8203;NOTE: The optical absorbance of the acid blank read against deionized water (reference) should be less than 0.015; the optical absorbance of the standard and samples should be between 0.100 and 1.000. For samples with very high or very low iron content, the volumes of acid extract (supernatant) and diH2O may need to be adjusted (Table 2): if absorbance is greater than 1.0, use a smaller sample (supernatant) volume; when absorbance is lower than 0.1, use a higher volume of the supernatant. Sample volume (Vsmp) is taken into account when calculating each sample&#39;s tissue iron content (see step 6).</li>
</ol>
6. Calculation of tissue iron content
<ol>
	<li>Calculate non-heme tissue iron content with the following equation: 
	Tissue iron (&#181;g/g dry tissue) = <img alt="Equation 1" src="/files/ftp_upload/63469/63469eq01v2.jpg" /> 
	AT = absorbance of test sample 
	AB = absorbance of acid blank 
	AS = absorbance of standard 
	Fes = iron concentration of WISS (&#181;g Fe/mL) 
	W = weight of dry tissue (g) 
	Vsmp = sample volume (variable volume of Supernatant in Table 2 converted to mL) 
	Vf = final volume of acid mixture after overnight incubation at 65 &#176;C (corresponding to acid volume plus dry tissue volume in mL; if sample weights do not differ significantly, assume a constant volume &#8776; 1 mL) 
	Vstd = iron standard volume (volume of WISS in Table 2 converted to mL) 
	Vrv = final reaction volume (Total volume in Table 2 converted to mL)</li>
</ol>

<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>

The authors have no conflicts of interest.

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><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 (&lt;em&gt;Mus musculus&lt;/em&gt;)</td><td>Charles River Laboratories</td><td></td><td></td></tr><tr><td>Carbonyl iron powder, &amp;ge;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 (&lt;em&gt;Dicentrarchus labrax&lt;/em&gt;)</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 ...

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

Research

JoVE Journal

Medicine

4.4K Views.  Universidade do Porto. Hier hebben we een protocol gegeven voor het meten van de niet-heemgehaltes in dierlijke weefsels. Met behulp van een eenvoudige gevestigde colorimetrische test die gemakkelijk kan worden geïmplementeerd in de meeste laboratoria. De Bathophenanthroline-Based Colorimetric Assay vereist geen geavanceerde vaardigheden of zeer dure apparatuur en kan worden aangepast aan microplaatformaat voor een hogere monsterdoorvoer en kosteneffectiviteit.Dit protocol is geschikt voor het detecteren va...

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

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Biochemistry

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as many of the common di-and trivalent cations used in IMAC resins have an inhibitory effect on enzymes. However, the extent of metal leaching and the impact of various eluting and reducing reagents are poorly understood in large part due to the absence of simple and practical transition metal quantification protocols that use equipment typically available in biochemistry labs. To address this problem, we have developed a protocol to quickly quantify the amount of metal contamination in samples prepared using IMAC as a purification step. The method uses hydroxynaphthol blue (HNB) as a colorimetric indicator for metal cation content in a sample solution and UV-Vis spectroscopy as a means to quantify the amount of metal present, into the nanomolar range, based on the change in the HNB spectrum at 647 nm. While metal content in a solution has historically been determined using atomic absorption spectroscopy or inductively coupled plasma techniques, these methods require specialized equipment and training outside the scope of a typical biochemistry laboratory. The method proposed here provides a simple and fast way for biochemists to determine the metal content of samples using existing equipment and knowledge without sacrificing accuracy.

We present an assay for easy quantification of metals introduced to samples prepared using immobilized metal affinity chromatography. The method uses hydroxynaphthol blue as the colorimetric metal indicator and a UV-Vis spectrophotometer as the detector.

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as ...

Chemistry

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS) for Quantification of Iron Redox Species (Fe(II), Fe(III))

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative conditions. Excessive iron results in free redox-active Fe(II) and can cause devastating effects within the cell like oxidative stress (OS) and death by lipid peroxidation known as ferroptosis (FPT). Therefore, quantitative measurements of ferrous (Fe(II)) and ferric (Fe(III)) iron rather than total Fe-determination is the key for closer insight into these detrimental processes. Since Fe(II)/(III) determinations can be hampered by fast redox-state shifts and low concentrations in relevant samples, like cerebrospinal fluid (CSF), methods should be available that analyze quickly and provide low limits of quantification (LOQ). Capillary electrophoresis (CE) offers the advantage of fast Fe(II)/Fe(III) separation and works without a stationary phase, which could interfere with the redox balance or cause analyte sticking. CE combined with inductively coupled plasma mass spectrometry (ICP-MS) as a detector offers further improvement of detection sensitivity and selectivity. The presented method uses 20 mM HCl as a background electrolyte and a voltage of +25 kV. Peak shapes and concentration detection limits are improved by conductivity-pH-stacking. For reduction of 56[ArO]+, ICP-MS was operated in the dynamic reaction cell (DRC) mode with NH3 as a reaction gas. The method achieves a limit of detection (LOD) of 3 &#181;g/L. Due to stacking, higher injection volumes were possible without hampering separation but improving LOD. Calibrations related to peak area were linear up to 150 &#181;g/L. Measurement precision was 2.2% (Fe(III)) to 3.5% (Fe(II)). Migration time precision was &#60;3% for both species, determined in 1:2 diluted lysates of human neuroblastoma (SH-SY5Y) cells. Recovery experiments with standard addition revealed accuracy of 97% Fe(III) and 105 % Fe(II). In real-life bio-samples like CSF, migration time can vary according to varying conductivity (i.e., salinity). Thus, peak identification is confirmed by standard addition.

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry CE-ICP-MS for Quantification of Iron Redox Species FeII, FeIII

This iron redox speciation method is based on capillary electrophoresis-inductively coupled plasma mass spectrometry with sample stacking combined with short analysis in one run. The method quickly analyzes and provides low limits of quantification for iron redox species across a diverse range of tissues and biofluid samples.

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative ...

Fluorescent Assay for Detection of Novel Iron Chelators

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Author Spotlight: Assessing the Impact of Novel Iron Chelators on Cancer Cell Metabolism

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

Cancer Research

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Biology

Assessing Iron Deposition in the Brains of 5xFAD Mice by Perls'/DAB Staining

Alzheimer's disease (AD), a common neurodegenerative disease, is the leading cause of dementia in the elderly. Iron deposition is closely linked to the pathogenesis of AD. In the AD brain, elevated iron levels are strongly correlated with characteristic AD pathologies, including &#946;-amyloid plaques, tau neurofibrillary tangles, and apoptosis. Increasing evidence suggests that oxidative stress, due to dysregulated iron metabolism, plays an important role in AD pathophysiology. Understanding the role of iron accumulation in the pathology of AD is, therefore, critical to treating AD. A comprehensive iron staining protocol is essential to detect iron expression and distribution in the brain. This protocol aims to describe a method to assess the quantity and distribution of iron deposition in the brain. We describe the processes involved in reagent preparation, mouse treatment, and preparation of brain sections. In addition, we present a detailed step-by-step protocol for histochemical iron detection in cerebral tissue sections, accompanied by a systematic analytical framework integrating both quantitative assessment and morphological evaluation to ensure a comprehensive interpretation of staining outcomes. This experimental protocol will help researchers understand iron depositions in the brain and support AD research.

This protocol presents methods for assessing the distribution and quantity of iron deposition in tissues, especially the brain. The protocol details the procedures for sample preparation, Perls/DAB staining, image capture, and data analysis.

Alzheimer's disease (AD), a common neurodegenerative disease, is the leading cause of dementia in the elderly. Iron deposition is closely linked to the ...

Neuroscience

Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes

Iron is essential for maternal and fetal health during pregnancy, with approximately 1 g of iron needed in humans to sustain a healthy pregnancy. Fetal iron endowment is entirely dependent on iron transfer across the placenta, and perturbations of this transfer can lead to adverse pregnancy outcomes. In mice, measurement of iron fluxes across the placenta traditionally relied on radioactive iron isotopes, a highly sensitive but burdensome approach. Stable iron isotopes (57Fe and 58Fe) offer a nonradioactive alternative for use in human pregnancy studies.
Under physiological conditions, transferrin-bound iron is the predominant form of iron taken up by the placenta. Thus, 58Fe-transferrin was prepared and injected intravenously in pregnant dams to directly assess placental iron transport and bypass maternal intestinal iron absorption as a confounding variable. Isotopic iron was quantitated in the placenta and mouse embryonic tissues by inductively coupled plasma mass spectrometry (ICP-MS). These methods can also be employed in other animal model systems of physiology or disease to quantify in vivo iron dynamics.

Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes

This article demonstrates how to prepare and administer transferrin-bound nonradioactive isotopic iron for studies of iron transport in mouse pregnancy. The approach for quantifying isotopic iron in fetoplacental compartments is also described.

Iron is essential for maternal and fetal health during pregnancy, with approximately 1 g of iron needed in humans to sustain a healthy pregnancy. Fetal ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...

Bathophenanthroline Sulfonate-Based Colorimetric Assay: A Simple and Rapid Method for Quantitation of Non-Heme Iron in Mouse Liver Tissue

Source: Duarte, T. L., Neves, J. V. <a href="https://www.jove.com/v/63469">Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay.</a> J. Vis. Exp. (2022)
In this video, we describe a colorimetric-based method to determine non-heme iron content in mouse liver tissue. Bathophenanthroline sulfonate is used as the chromogenic substrate in the presence of a reducing agent, which converts ferric iron to chelatable ferrous iron.

Source: Duarte, T. L., Neves, J. V. Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay. J. Vis. Exp. (2022)
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Caco-2 Cell Bioassay: An In Vitro Method for Measuring Iron Bioavailability in Complex Food Sources

Source: Glahn, R. P. <a href="https://app.jove.com/v/63859">The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability.</a> J. Vis. Exp. (2022)
This video demonstrates an in vitro technique to assess iron bioavailability in complex food sources. The technique combines the simulation of human digestion along with iron uptake by differentiated Caco-2 cells. Intracellular ferritin formation acts as a marker for cellular iron uptake.

Source: Glahn, R. P. The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability. J. Vis. Exp. (2022)
This video demonstrates an in vitro ...

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

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