Sulfide silver autometallography to differentiate the ultrastructural localization of iron-carbohydrate complexes inside macrophages

Iron is an essential element for numerous physiological processes in the human body, and maintaining proper iron homeostasis is critical for health. Iron imbalance can lead to conditions like iron deficiency anemia (IDA). Intravenous (IV) iron drugs, including iron-carbohydrate complexes such as iron sucrose (IS) and ferric carboxymaltose (FCM), are commonly used in the treatment of IDA. However, the cellular mechanisms underlying the uptake and intracellular fate of these complexes remain poorly understood despite over seven decades of clinical use. This study introduces a novel application of sulfide silver autometallography (ssAMG) to track iron inside macrophages treated with two widely used intravenous iron products, IS and FCM. Using this technique, the ultrastructural localization of iron in macrophages was visualized in comparison to ferric ion (Fe³⁺). ssAMG improved the visualization of IS and FCM, revealed as crystalline, cluster-like particles, i.e. silver precipitates, localized inside intracellular vesicles. Interestingly, for both iron-carbohydrate complexes, the silver precipitates were observed exclusively inside the cells and not on the cell surface as seen for ferric ion. These results suggest that IS and FCM are not internalized by macrophages as ferric iron bound to transferrin and provide new insights into the cellular processing of iron-carbohydrate complexes to advance the understanding of iron homeostasis.