Here we have provided a protocol for measuring the non-Heme contents in animal tissues. Using a simple well-established colorimetric assay that can be implemented easily in most laboratories. The Bathophenanthroline-Based Colorimetric Assay does not require sophisticated skills or highly expensive equipment and can be adapted to microplate format for higher sample throughput and cost effectiveness.
This protocol is suited for detecting alterations in tissue iron levels in a variety of experimental animal models of iron overloads or iron efficiency. Begin by cutting a tissue sample weighing 10 to 100 grams with a scalpel blade. Weigh it accurately in an analytical balance over a small piece of parafilm.
Use plastic tweezers to place the piece of tissue in a 24 well plate. Let it dry on a standard incubator at 65 degree celsius for 48 hours. Alternatively place the wade piece of tissue using stick tweezers in an iron free Teflon cup and dry it in the laboratory microwave digestion oven.
Place each dried piece of tissue over a small piece of paraform inside an analytical balance and weigh it accurately. Transfer each dried piece of tissue into a 1.5 milliliter micro centrifuge tube. Add a lit 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 micro pipette fitted with plastic tips to transfer 500 microliters of the clear acid extract into a new 1.5 milliliter micro centrifuge tube.
Prepare chromogen reactions directly into the flat bottom 96-well, clear untreated polystyrene micro plates. Incubated room temperature for 15 minutes. Measure sample absorbance in a plate reader at a wavelength of 535 nano meters against a deionized water reference.
Determination of non-heme iron by the cuvette and 96-well microplate based colorimetric assays are shown here. The correlation between cuvette based and micro plate based non-heme iron levels in 55 tissue samples from six month old male black, six mice, including liver, spleen, heart, lungs and bone marrow are shown here. The high correlation between the two methodologies indicates that the micro plate based method is a valid alternative to the original cuvette based method.
Finally, non-heme iron levels were quantified with the micro plate based method after acidic digestion of sample derived from the same tissues with either a mixture of hydrochloric acid and trichloroacetic acid or hydrochloric acid alone. The high correlation shows that trichloroacetic acid can be emitted from the acid digestion. The representative image shows the non-heme iron levels in the liver, spleen, heart and pancreas of male black six wild type mice and male hepcidin knockout mice on black six genetic background measured with Bathophenanthroline-Based Colorimetric Assay.
The disruption of hepcidin leads to a hemochromatosis like iron deposition phenotype with severe hepatic, pancreatic and cardiac iron accumulation and splenic iron depletion. Hepatic non-heme iron levels in juvenile female European sea bass following experimental iron modulation are shown here. Hepatic iron levels increased massively in iron treated animals.
Whereas anemia caused a mild reduction in the hepatic iron stores. The representative images show the non-heme iron levels in one month old Drosophila measured with a Bathophenanthroline-Based Colorimetric Assay. Results show the non-heme iron content in each pool of 20 flies or the estimated iron content of each fly.
Considering an average weight of 0.64 milligrams per fly. While attempting this procedure remember to thoroughly clean all the glass wear and do not allow the metallic laboratory materials to come into contact with any reagent or solution due to the risk of contamination.
