Malnutrition contributes to half of all global child deaths. Severe malnutrition interferes with the liver’s synthesis of albumin, complement and coagulation factors, and bile acids (BAs). BA deficiency impairs nutrient absorption and growth and alters signaling through nuclear receptors including farnesoid-X-receptor (FXR) to impact a wide range of processes. Using a mouse model of early postnatal malnutrition, we reported that decreased BA synthesis in malnutrition causes decreased FXR activation and decreased expression of FXR target genes including coagulation factors. The resulting malnutrition-induced coagulopathy can cause child mortality. It is not known why BA synthesis is impaired in malnutrition. Our published data and new preliminary data indicate malnutrition impairs the activity (not expression) of the rate-determining enzyme (CYP7A1) in the classic pathway of BA synthesis due to depletion of the essential cofactor heme. We present novel evidence that restoring heme increases BA synthesis. We now seek to understand why heme synthesis is impaired in malnutrition. Heme is generated from tricarboxylic acid (TCA) cycle products and enzymes that require iron- sulfur (Fe-S) clusters for stability. Fe-S clusters are derived from both Fe and S-containing amino acids (AAs), which are found in dietary protein or generated by transsulfuration. Like most low-protein diets, our mouse malnourishing diet is deficient in S-containing AAs. The mice exhibit decreased expression of transsulfuration and Fe-S cluster-dependent TCA cycle enzymes, suggesting that deficiency of S-containing AAs and Fe-S clusters drives liver dysfunction in malnutrition. Indeed, we decreased BA synthesis by 50% by maintaining hepatocytes in low-AA media, and we restored BA production by adding S-containing AAs (but not other AAs). Thus, we hypothesize that deficiency of S-containing AAs in malnutrition impairs BA synthesis by disrupting TCA cycle function, heme biosynthesis, and CYP7A1 activity. Our Specific Aims are to 1) Characterize in AA- deficient hepatocytes TCA cycle dysfunction by quantifying flux of labeled glucose through the TCA cycle and measuring expression levels and activity of Fe-S cluster-dependent TCA cycle enzymes before and after heme treatment; 2) Define the role of S-containing AAs and heme in BA synthesis by measuring BA synthesis by hepatocytes maintained in low-AA media and treated in a high-throughput manner with combinations of AAs and heme and using adeno-associated virus or siRNA to manipulate levels of transsulfuration and Fe-S cluster- dependent enzymes; and 3) Determine whether a novel nutritional therapeutic can rescue BA synthesis in malnutrition by feeding mice the malnourishing diet supplemented by S-containing AAs, then quantifying TCA cycle function, heme synthesis, and BA production in primary hepatocytes. Expected outcomes include elucidation of a novel link between malnutrition, TCA cycle and heme homeostasis, and BA synthesis. The r...