The importance of elucidating the mechanism(s) involved in iron metabolism is of immense significance in studying hematological diseases. The pathophysiological significance of oxygen free radicals and transition metal catalysis extends to any organ system and includes carcinogenesis including leukemia, coronary artery disease, inflammation, aging and neurodegenerative disease and stroke. An important aspect of these studies is the development of iron defined reagents to elucidate in-vitro mechanism(s) of iron metabolism. The approach employed in the Phase I effort was to adapt cell lines, Raji and IEC-6, to serum free reagent, then sequentially replace each ill-defined component of the serum-free reagent with chemically defined iron-free reagents. We have accomplished replacing one of the most ill-defined components, BSA with a mixture of linoleic acid, oleic acid and cholesterol bound to the water solubilizing agent, B-cyclodextrin. The transferrin was replaced with iron defined chemicals. With the replacement of these ill-defined components the Applicants and poised to answer significant questions in the Phase II proposal: 1) The optimization of these components for the in-vitro proliferation of Raji, IEC-6 and K562 cells; 2) development of an iron defined reagent that supports primary cultures of rat duodenal cells; 3) the use of such media in iron metabolism studies.
