Folates are required for DNA synthesis, cell regeneration, and methionine metabolism. Folate deficiency results in anemia, functional abnormalities of the small intestine, and hyperhomocysteinemia with increased risk of vascular disease. This application proposes a systematic study of the membrane proteins that may regulate folate absorption in the intestine and folate uptake by the liver and the proximal renal tubule of the pig and their single and synergistic perturbations by folate deficiency and chronic alcohol consumption.
The specific aims are: 1) to establish the molecular sequences, tissue expression, and kinetics of three folate regulatory proteins: jejunal brush border folate polyglutamate hydrolase (BBFH) and the liver and kidney membrane folate binding protein (FBP) and reduced folate carrier (RFC); 2) to use human tissues to evaluate the relevance of the pig as a model for human folate homeostasis; and 3) evaluate perturbations of these proteins by combinations of folate deficiency and chronic alcohol consumption in micropigs. The distribution of folate coenzyme forms will be determine in liver, kidney, and intestinal mucosa and in urine. The PI will isolate full length clones for the BBFH and RFC from intestine from pig and humans. Antibodies to the expressed proteins will be obtained. The cDNA's and antibodies will be used to study the effect of alcohol and folate deficiency on transcription and expression of these proteins. Isolated membrane vesicles will be used to study fatty acid composition and for functional studies of folate binding and transport in normal, folate deficient, and folate deficient alcoholic pigs. The studies are expected to form a basis for future studies on gene regulation of each of the membrane proteins and on a molecular understanding of the pathogenesis of folate deficiency in intestinal diseases.
