Why does acid digestion matter for non-heme iron quantitation?

Acid digestion with hydrochloric and trichloroacetic acids releases non-heme iron from proteins while leaving heme iron intact, enabling specific quantification of the non-heme fraction.

How does thioglycolic acid improve assay specificity?

Thioglycolic acid reduces ferric iron to the ferrous state for chelation with BPS and prevents interference from divalent cations like copper that could otherwise bind the chromogen.

What does absorbance measurement at 535 nm indicate?

Absorbance at 535 nm corresponds to the formation of the ferrous-BPS complex, providing a direct, quantitative readout of non-heme iron concentration in the sample.

Why is a 15-minute incubation sufficient for color development?

The 15-minute room-temperature incubation allows complete chelation of ferrous iron by BPS to form the stable pink complex, ensuring consistent signal generation.

What role does the acid blank play in assay accuracy?

The acid blank, prepared without tissue, accounts for background absorbance from reagents, enabling accurate subtraction and true signal measurement from the sample.

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

JoVE Encyclopedia of Experiments

Biological Techniques

0 views • 3:03 min • July 8th, 2025

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In tissue, iron, an essential mineral crucial for various metabolic processes, normally exists sequestered to hemoproteins and non-heme iron proteins. Hemoproteins include ferrous or heme iron bound to porphyrin rings, whereas non-heme iron proteins lack the porphyrin ring structure and mostly comprise ferric or non-heme iron.

To estimate non-heme iron content in mouse liver tissue, treat a dried, harvested mouse liver tissue fragment with low concentrations of hydrochloric acid and trichloroacetic acid. Incubate at high temperatures to digest the tissue and facilitate acid-induced protein precipitation.

Owing to low acid concentrations, non-heme iron gets released from the associated proteins. Heme iron, being bound to the porphyrin ring, remains unaffected.

After cooling, transfer the desired volume of supernatant containing non-heme iron into a multi-well plate. Add the chromogenic iron-chelator, bathophenanthroline sulfonate, BPS, supplemented with thioglycolic acid, a reducing agent, in suitable buffer and incubate.

Thioglycolic acid reduces all the ferric non-heme iron to ferrous iron, which can readily bind to BPS in a 1:3 ratio, forming a highly stable, pink-colored complex. Additionally, thioglycolic acid prevents interference due to any contaminating divalent metal cations such as copper that may interact with BPS.

Place the multi-well plate in a microplate reader to measure the absorbance of the pink ferrous-BPS complexes, representative of non-heme iron concentration in mouse liver tissue fragment.

Transfer each dried piece of tissue into a 1.5-milliliter microcentrifuge tube. Add 1 milliliter 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 micropipette fitted with plastic tips to transfer 500 microliters of the clear acid extract into a new 1.5-milliliter microcentrifuge tube.

Prepare chromogen reactions directly into the flat-bottom, 96-well, clear, untreated, polystyrene microplates. Incubate at room temperature for 15 minutes.

Measure sample absorbance in a plate reader at a wavelength of 535 nanometers against a deionized water reference.