Ferrozine-Based Iron Quantification of Iron Overloaded Mice: a Simple, Robust Method for the Assessment of Iron Chelation Therapies
Authors: Tran David T , Petersen Max J , Noble Charles O , Hayes Mark E , Working Peter , Szoka Francis C 
Published: October 2019

Abstract
Introduction: We describe a low heat nitric acid digestion and colorimetric ferrozine-based iron assay that provides a fast, inexpensive and accurate alternative to high temperature animal tissue processing and inductively coupled plasma mass spectrometry (ICP). This technique is useful for the quantification of iron in iron overloaded animal models and diseases such as β-thalassemia, sickle cell anemia, and myelodysplastic syndromes. We applied it to evaluate iron removal by iron chelation therapies such as liposome-encapsulated deferoxamine (LDFO).
Methods: CF-1 mouse liver, spleen, heart, plasma, urine and feces were digested in nitric acid (70%) at 65 °C for 1-2 hours. For large tissues such as the liver, tissues were sectioned at 50 mg fractions (n = 4) to also assess iron homogeneousness. Digested samples were bleached with hydrogen peroxide (30%) and diluted with water before iron quantification by ICP or the ferrozine-based assay. For the ferrozine-based assay, nitric acid was neutralized with ammonium acetate and iron was reduced with ascorbic acid before reaction with ferrozine for the colorimetric assessment of the ferrozine-ferrous iron complex at 550 nm. For iron overloaded models, CF-1 mice (n = 4) were loaded I.V. with iron dextran, iron sucrose, liposome encapsulated iron, or horse ferritin for over a week before sacrifice. Iron removal studies of tissues and excreta from iron overloaded CF-1 (n = 4) started 2 weeks after iron dextran overloading. Animals were dosed with either deferoxamine (DFO) and LDFO by I.V. and Exjade by oral gavage. Urine and feces were collected daily at the start of treatment. Animals were sacrificed 7 days post treatment. Tissues and excrements were digested and measured for iron by the ferrozine-based assay.
Results: The ferrozine-based tissue iron quantification assay yields high iron recovery from mouse tissue. CF-1 liver spiked with iron dextran or horse ferritin had an iron recovery of 99±2% and 97±2%, respectively. Liver iron measurements of iron dextran overloaded mouse models resulted in identical iron measurements for the ferrozine-based assay and ICP. For CF-1 mice treated with iron dextran I.V. (0, 100, 300, 600 mg/kg, n = 4), liver iron is highly correlative between the two iron quantification techniques with a slope of 1.03 and R2 of 0.999. In addition, there is a linear iron overloading effect in both the liver (R2 = 0.99) and spleen (R2 = 0.98). CF-1 mice were also iron overloaded with iron sucrose, liposome encapsulated iron, and horse ferritin with dose dependent tissue overload. For all iron overloaded models tested, liver iron and spleen iron were homogenous per tissue when multiple sections were analyzed with an average low 6% difference in iron content. Despite high liver and spleen overloading, none of these iron carriers resulted in statistically significant heart iron overload. Iron dextran overloaded CF-1 mice (100 mg/kg) were treated with LDFO, DFO, and Exjade. LDFO at 100 mg/kg I.V. greatly reduces iron levels in the liver (p = 0.019) and spleen (p = 0.014) compared to non-effective no treatment, free DFO (p = 0.3), and empty liposomes (p = 0.1). Exjade at 30 mg/kg by oral gavage did not result in statistically significant iron removal in the liver or spleen (p < 0.2). Over the first four days, urine and feces were collected daily and also analyzed for iron. Results revealed that iron clearance by LDFO is primarily in the urine (p = 0.022 urine; p = 0.8 feces) while Exjade removed iron appeared in the feces (p = 0.06 feces; p = 0.013 urine). During this short period, drug efficiency in iron excretion (5%) from one dose of the novel LDFO at 100 mg/kg was equivalent to four daily doses of the Exjade at 50 mg/kg/dose.
Conclusion: The low heat nitric acid digestion and ferrozine-based tissue iron quantification assay is a simple, precise, highly reproducible tool for the assessment of tissue and excretion iron. The assay enabled the rapid, low cost evaluation of novel iron chelation therapies. We gratefully acknowledge support by NIH SBIR Phase 1 Grant 1R43HD075429-01 and NIH SBIR Phase 2 Grant 2R44HD075429-02.

Disclosures
Tran: Zoneone Pharma, Inc.: Employment. Petersen:Zoneone Pharma, Inc.: Employment. Noble:Zoneone Pharma, Inc.: Employment, Equity Ownership. Hayes:Zoneone Pharma, Inc.: Employment, Equity Ownership. Working:Zoneone Pharma, Inc.: Consultancy, Equity Ownership. Szoka:Zoneone Pharma, Inc.: Consultancy, Equity Ownership.