Project Summary Iron deficiency is the single most common nutrient deficiency in the world impacting one-third of the world’s population. Recent analysis of the US National Health and Nutrition Examination Survey (NHANES) and Supplemental Nutrition Program for Women, Infants, and Children (WIC) data established dietary iron intakes are decreasing in the US population and the decline parallels increasing rates of anemia. Increases in iron deficiency may be caused by declining diet quality and/or high rates of nutrition and food insecurity. Iron deficiency is the leading cause of anemia which severely impacts physical and cognitive development, work capacity, and leads to poor health outcomes. The WHO categorizes iron-deficiency anemia as hemoglobin levels <120 mg/dL (non-pregnant females) and iron deficiency as ferritin levels <15 ng/mL in individuals over 5 years old and <12 ng/mL in children under 5 years old. These ferritin levels, which are used clinically, are not agreed upon. First, a level of 30 ng/mL has been suggested to have 92% sensitivity and 98% specificity for correlating iron deficiency with the absence of iron stores in the bone marrow. Second, marginal iron depletion (normal hemoglobin levels with inadequate ferritin stores, defined as ferritin levels between 15-30 ng/mL) is suggested to impair cellular functions. In human studies, individuals with marginal iron depletion have reduced physical work capacity, yet their ability to transport oxygen is not impaired. This indicates other pathways involved in physical work capacity may be compromised, yet these cellular pathways and their mechanisms have yet to be determined. Furthermore, the correlation between iron-dependent tissues and cellular compartments, blood biomarkers of iron levels, and the cellular pathways that lead to reduced work capacity have not been determined. Our preliminary data indicate that impairing iron status from mild to severe depletion in mouse myoblast cells reduces cellular respiration and the contribution of ATP from mitochondrial oxidative phosphorylation before the cellular biomarker (transferrin receptor) for iron deficiency responds. Our overall objectives in this application are to 1) establish the relationship between blood values of ferritin and hemoglobin with tissue and cellular compartment levels of iron, and 2) determine the relationship between iron status and iron-dependent mitochondrial and cellular functional declines. Our central hypothesis is that (1) certain tissues and cellular compartments are more susceptible to iron loss and deplete at different rates, occurring before blood biomarkers/levels of iron decline, and (2) mitochondrial function is compromised with marginal iron depletion and is an underlying cause of skeletal muscle reduced work capacity. We will test this hypothesis in two specific aims. In the first aim, we will use an established mouse model and dietary iron intervention to alter iron status to determine the associatio...