Iron is found in many enzymes as an important constituent element. All iron in blood is bound to transferrin and is transported to erythroblasts and various tissues for synthesis of globin proteins that require iron, such as myoglobin and hemoglobin. Iron is essential for the production of proteins that transport oxygen, and its deficiency can cause iron deficiency anemia, chronic hemorrhagic anemia, and infectious anemia. Increased transferrin and high concentrations of iron are also observed in hepatitis and cirrhosis. Aplastic anemia and pernicious anemia also show increased iron.

This method determines iron concentration by observing the color change in the visible region due to the formation of a chelate complex between ferrozine and iron.

Iron bound to transport proteins such as transferrin is dissociated by a weak acid or denaturing agent in the reagent to form an iron-ferrozine complex. The iron concentration can be determined by measuring this complex at a wavelength of 560 nm.

◯ Samples that can be directly quantified with this kit

Examples: serum, plasma, urine

The denaturing agent in the kit dissociates iron from protein and causes coloration. No special pretreatment is required.

※ Do not use blood collection tubes containing EDTA which may affect the assay value.

※ When using urine samples, preserve it by adding HCl and adjust the pH to 2~3 when measuring.

◯ Samples that require acid extraction

Examples: cell lysates, tissue homogenates, and other aqueous solutions

By lowering the pH of the sample, iron is dissociated from the protein, and the supernatant is collected and used as the assay sample. However, only the following chemicals can be measured.

・Free iron

・Protein-bound (coordinated) iron (Protein-Fe2+, e.g. transferrin, ferritin, metallothionein)

・Other coordinated iron

※ Lysis buffer containing EDTA should not be used because it may affect the assay value.

※ Organic iron and heme iron should be acid decomposed first.

◯ Samples that require microwave method and decomposition of organic matter with mixed acid.

Examples: Samples containing strong chelating agents such as EDTA, organic iron (-C-Fe-C- single bonded), and iron encapsulating cyclic ligands (Fe-porphyrin, etc.)

It is necessary to measure chemical species that do not dissociate by acid extraction or samples containing interfering substances such as strong iron chelating agents.

※ After decomposition of organic matter by each decomposition method, adjust the pH to 2 to 3, and use a constant volume sample as the assay sample.

(1) Add 40 µl of DIW, standard solution, and assay sample to wells of 96-well plate.

(2) Apply 200 µl of RA to each well.

(3) Incubate the plate at room temperature for 5 minutes.

(4) Measure absorbance OD1 at the wavelength of 560 nm.

(5) Add 8 µl of chromogenic solution to each well.

(6) Incubate the plate at room temperature for 5 minutes.

(7) Measure absorbance OD2 at the wavelength of 560 nm.

(8) Calculate the iron concentration using the following formula:

(1) Measurement of control serum with this kit

Figure 1: Results of measurement of control serum with known iron concentration.

(2) Measurement of iron concentration in pig liver using this kit

Figure 2: Results of pig liver iron concentration measurement using the ICP-OES method and this kit

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Iron Assay kit LS – Ferrozine method | Metallo Assay LS

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