We are evaluating major sequelae of excessive hemoglobin catabolism which follow episodes of hemolysis or repeated blood transfusions. These include high serum heme levels, increased hepatic heme uptake and iron accumulation. Two serum proteins bind heme entering the circulation. It is initially bound by albumin and subsequently by hemopexin (Hx) which transports it to the liver, the Hx being recycled and heme being reutilized and degraded. We are studying the dependence of the kinetics of heme transfer from albumin to Hx on competing anions and are probing into the nature of Hx's heme-binding site. The major emphasis of the proposed work is to identify factor(s) and mechanisms, which control Hx synthesis. In particular we will investigate the interrelationship of iron and heme metabolism in the regulation of Hx metabolism. These studies are suggested because we have shown that exogenous heme, depending on the amount entering hepatocytes, changes the rate of Hx synthesis, and desferrioxamine, an iron chelator, substantially increases Hx levels in vivo and in isolated perfused livers. The hypothesis that the relative concentrations of iron and/or heme in one or more subcellular compartments is responsible for the regulation of Hx synthesis will be studied in experiments in vitro and in vivo. Several hepatocyte culture systems will be used for determining whether it is iron and/or heme or additional factors which affect Hx synthesis. Whether this regulation is exerted pre- or post-translationally will be determined employing rabbit reticulocyte lysates and liver mRNA isolation from animals in various deranged states of heme and/or iron metabolism. Subsequent cellular events, such as glycosylation will also be assessed. Significancant results will be evaluated in rats in vivo and will be correlated in patients will diseases of aberrant iron and/or heme metabolism. Preliminary results in Beta-thalassemia suggest that Hx levels approach normal during chelation therapy, a finding providing strong evidence that iron as well as heme metabolism may regulate Hx synthesis.
