The overall objective of this proposal is to characterize the manganese binding ligands in milk and to study their functions in absorption of this metal. Using extrinsic labvel techniques several manganese binding ligands have been identified in human milk, cow's milk and infant formula by the investigator. These manganese binding proteins and low molecular weight lighand will be further purified and characterized using biochemical techniques such as gel permeation and ion exchange column chromatography, disc and horizontal polyacrylamide gel electrophoresis with and withour SDS, analytical and preparative isoelectric focusing and thin layer chromatography. The physicobiochemical properties, including the number of metal binding sites, metal binding affinities, amino acid composition, and if time allows, the primary seqeunce of the peptidic species will be investigated. The similarity and difference between endogenous binding ligands and extrinsically labelled binding ligands will be studied by using in vivo and in vitro labelling of mild from rats. The presence of manganese binding ligands in the intestinal mucosa of adult and suckling rats and in cultured skin fibroblasts will also be investigated. their properties will be compared with those of the milk metal carriers. The relative bioavailability of the mineral from human breast milk, cow's milk and infant formula, and the effects of the chemical form on bioavailability, such as when they are present as inorganic salts, organic salts, or metallopeptides will be studied at three different levels, namely, whole animal level using suckling rats, tissue level using cultured rat intestine and cellular level using cultured skin bibroblasts. Radioactive Mn will be used, and its uptake will be measured by counting the isotopic content of the suckling rat, tissue or cells. the kinetics of the uptake process for Mn from extrinsic labelled milk will be studies at all three levels. The effects of metabolic and prtein synthesis inhibitors, and temperature will be investigated using the two in vitro systems. One of the long term objectives of this proposal is to understand the effect of chemical environment on the bioavailability of the trace minerals. The information obtained from this project will help to clarify the mechanism of absorption of manganese. This knowledge will insure the production of a more approporate substitue for breast mild and will caution the development of trace mineral deficiency or toxicity in infants. The procedures developed will be useful for more vigorous comparison among the different breast mild substitutes. They can also be used to evaluate the bioavailability of other essential trace minerals, vitamins, etc. in milk. This study will also provide information concerning the maturation process of the gastrointestinal tract.
