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 ov....

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 ....

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