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

Summary

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.

Abstract

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.

Introduction

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 worldwide^1,2,3.

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 ago^5,6. 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.

Protocol

C57BL/6 mice were commercially purchased and hepcidin-null (Hamp1^−/−) 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, Direção-Geral de Alimentação e Veterinária (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.

1. 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.
2. 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.
3. 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 µL of deionized water and 10 µL 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.
4. 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.
5. 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 µL 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.
6. Working Iron Standard Solution (WISS): Add 13.5 µL of 37% hydrochloric acid to 500 µL of deionized water. Add 10 µL of the Stock Iron Standard Solution and make up the final volume to 1 mL with deionized water (11.169 µg of Fe/mL, 200 µM; measured by AAS).
   ​NOTE: The working solution should be prepared freshly on the day of use.

2. Sample drying

1. 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).
2. 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 °C for 48 h.
3. 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'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.
4. 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).

3. Sample acidic digestion

1. Using plastic tweezers, transfer each dried piece of tissue into a 1.5 mL microcentrifuge tube.
2. 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.
3. Digest the tissues by incubating the microcentrifuge tubes in an incubator at 65 °C for 20 h.
4. After cooling to room temperature, transfer 500 µL 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.
   ​NOTE: At this point, the acid extracts can be immediately used for the colorimetric assay or frozen at -20 °C for later use. Completely thaw frozen samples to room temperature and vortex them prior to use.

4. Color development

1. 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.
2. Incubate at room temperature for 15 min.

5. Absorbance reading

1. 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.
   ​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 (V_smp) is taken into account when calculating each sample's tissue iron content (see step 6).

6. Calculation of tissue iron content

1. Calculate non-heme tissue iron content with the following equation:
   Tissue iron (µg/g dry tissue) =
   A_T = absorbance of test sample
   A_B = absorbance of acid blank
   A_S = absorbance of standard
   Fe_s = iron concentration of WISS (µg Fe/mL)
   W = weight of dry tissue (g)
   V_smp = sample volume (variable volume of Supernatant in Table 2 converted to mL)
   V_f = final volume of acid mixture after overnight incubation at 65 °C (corresponding to acid volume plus dry tissue volume in mL; if sample weights do not differ significantly, assume a constant volume ≈ 1 mL)
   V_std = iron standard volume (volume of WISS in Table 2 converted to mL)
   V_rv = final reaction volume (Total volume in Table 2 converted to mL)

Results

Cuvette versus 96-well microplate comparison
The measurement of tissue non-heme iron by reaction with a bathophenanthroline reagent originally described by Torrance and Bothwell^5,6 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...

Discussion

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 Bothwell^5,6. 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...

Materials

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