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

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.