The development of the ferene-based iron assay

A modified ferene assay was developed to analyze the labile and total iron content by manipulating ascorbic acid concentration. This section outlines three subsequent methods for the development of this assay, (i) determining iron concentrations in buffer conditions, (ii) distinguishing total and labile iron in cell lysates, and (iii) distinguishing labile and chelatable iron in cell lysates.

Three analytes were prepared with desired concentrations in distilled water;

(Fe) 100 μM of free iron.

(DFO-Fe) 100 μM of iron pre-chelated with 2 mM deferoxamine. 100 μM of iron solution in distilled water was incubated with 2 mM DFO for 48 h.

(DFO) 2 mM free deferoxamine.

Nine working solutions were prepared, each with 5 mM ferene in ammonium acetate buffer (pH 4.5, 2.5 M) with varying concentration of ascorbic acid (0, 1, 5, 10, 25, 50 100, 250, and 1000 mM). All the solutions were filtered (0.2 μm PVDF syringe filter) before use.

Iron content of the three analytes was measured in nine different sets of working solutions. A fresh calibration curve was generated using iron standards ranging from 0 to 400 μM (as outlined in 2.2.) for each set of working solution. This assay has been conveniently outlined in Supplementary Table ².

Each set has 100 μL of analyte (either Fe, DFO-Fe, or DFO) and iron standards (eight samples) in separate Eppendorf tubes. To each Eppendorf tube, 100 μL of ammonium acetate buffer (pH 4.5, 2.5 M) and 120 μL working solution were added. The resultant solution was vortexed and left overnight at room temperature. Absorbance was measured as described earlier in “Absorbance measurements”.

Iron concentrations in different analytes in the presence of varying ascorbic acid concentrations were determined by interpolating from the standard curve generated using iron standards (Supplementary Fig. ^S1). Calibration curves were generated for nine different ascorbic acid concentrations.

Iron overload HepG2 cell lysates were prepared as outlined previously. We define labile iron as iron chelated and detected by ferene using a working solution with a specific low ascorbic concentration (10 mM for final assay-see “Labile iron measurements using the u-ferene assay”) from undigested samples. Similarly, total iron is defined as iron chelated and detected by ferene using a working solution with a particular high ascorbic concentration (1 M for final assay-see “Total iron measurements using the u-ferene assay”) from nitric acid digested samples. In order to distinguish labile and total iron, different working solutions with varying ascorbic acid concentrations were investigated.

A set of four working solutions were prepared, each with 5 mM ferene in ammonium acetate buffer (pH 4.5, 2.5 M) with four varying ascorbic acid concentrations (10, 50, 250, and 1000 mM). For labile iron measurements, 100 μL of as made cell lysates were transferred into separate Eppendorf tubes. For total iron measurements, 100 μL of nitric acid digested cell lysates (as outlined in 2.5) were aliquoted into separate Eppendorf tubes. Iron standards (100 μL) were also aliquoted into separate Eppendorf tubes.

Ammonium acetate buffer (pH 4.5, 2.5 M) (100 μL) and 120 μL of working solution were added to all tubes. The samples were vortexed and left overnight at room temperature. The absorbance was measured in 200 μL of the resultant solution, as outlined in “Absorbance measurements”.

Cell lysates for both non-iron treated and iron overloaded HepG2 cells were prepared as outlined in “Cell culture and treatments”. For the purpose of this investigation, chelatable iron is defined as the portion of labile iron that is chelated by iron chelators and subsequently prevents the ferene-based detection of iron using a working solution at a particular low concentration of ascorbic acid.

Deferoxamine (DFO), deferiprone (DFP), and deferasirox (DFX) (50 μM each) were prepared in PBS. Iron overloaded cell lysates were treated with chelators; 250 μL of iron loaded HepG2 cell lysates were transferred into a clean Eppendorf tube followed by 50 μL of 50 μM chelator—either DFO, DFP or DFX. As negative controls, 250 μL of non-iron loaded and 250 μL iron loaded HepG2 cell lysates were also transferred into clean Eppendorf tubes followed by 50 μL of PBS only. These samples were left overnight at room temperature.

For labile iron: Working solution was prepared with 5 mM ferene in ammonium acetate buffer (pH 4.5, 2.5 M) with 10 mM of ascorbic acid. Cell lysates and iron standards (100 μL each) were transferred into separate Eppendorf tubes. 100 μL of ammonium acetate buffer (pH 4.5, 2.5 M) and 120 μL of working solution were added to each tube.

For total iron: Working solution was prepared with 5 mM ferene in ammonium acetate buffer (pH 4.5, 2.5 M) with 1 M of ascorbic acid. Acid digested cell lysates (100 μL) (as outlined in 2.5) and iron standards were transferred into separate Eppendorf tubes. Ammonium acetate buffer (100 μL) (pH 4.5, 2.5 M) and 120 μL working solution were added to each tube.

For iron quantification: These tubes were vortexed and left overnight. Absorbance was measured as described earlier in “Absorbance measurements”.