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Iron is an essential element for the proper functioning of the human body, and its homeostasis requires careful regulation. This regulation is performed primarily by hepcidin, a hormone secreted by the liver that controls the flow of iron within the body. It acts as a hyposideremic factor, reducing the expression of ferroportin, the only transmembrane protein currently known to export iron into the extracellular environment. This has the effect of decreasing intestinal absorption and increasing intracellular retention, particularly in macrophages. Hepcidin is mainly stimulated by the elevation of iron and inflammation while the activation of erythropoiesis inhibits it. Understanding its regulation allows a better understanding of the pathophysiological mechanisms of overload diseases and iron deficiency. Therefore, hepcidin analysis is interesting for the exploration of iron homeostasis. Hepcidin analysis can be used, in combination with other biological parameters of iron status, to improve treatment of iron metabolism disorders. Thus, a liquid chromatography–mass spectrometry technique was implemented at Grenoble University Hospital; the analytical performance of this assay met the laboratory requirements in terms of analysis reliability and robustness.