The long-term goal of this project is to determine the human requirement for biotin.
The specific aims are to determine the validity of newly developed indices of biotin nutritional status in both animal and clinical studies, to determine the chemical nature of avidin-binding substances recently discovered in human plasma and milk, and to determine biotin nutritional status longitudinally in pregnancy and in infancy. The studies are important because several lines of evidence suggest that biotin nutritional status is inadequate in some individuals in the populations to be studied. The studies are timely because more sensitive, chemically specific assays have demonstrated that 1) plasma biotin levels are low in patients with symptomatic biotin deficiency, contrary to results of the O. danica bioassay, 2) a substantial percentage of the avidin-binding substances in human plasma and milk is not biotin, contrary to previous assumptions, and 3) the 3 to 5 fold increase in plasma and milk content of """"""""biotin"""""""" that follows acid hydrolysis is not due to release of biotin from protein, contrary to previous assumptions. Previous studies provide evidence that increased urinary excretion of 3-hydroxyisovaleric acid (3-HIV) reflects impaired leucine metabolism as a consequence of deficient activity of the biotin-dependent enzyme 3- methylcrotonyl-CoA carboxylase and that increased percentage composition of the odd-chain fatty acids 15:0 and 17:0 in plasma and tissue phospholipid reflect deficient activity of the biotin- dependent enzyme propionyl-CoA carboxylase. Using 1) an 125I- avidin/HPLC assay for biotin in blood, urine, and tissue, 2) standard assays for the biotin-dependent carboxylases in tissues 3) GC/MS measurements of 3-HIV and 4) GC to measure fatty acid composition of plasma phospholipid, experiments will be conducted in the biotin deficient rat to confirm that 3-HIV excretion is an early sensitive indicator of tissue biotin depletion, to determine the effects of leucine intake and feeding/fasting on 3-HIV excretion, and to access the contribution of bacterial synthesis to absorbed biotin. Using several of these same indices of biotin nutritional status, we propose to determine whether plasma and urinary biotin decline longitudinally during total parenteral alimentation, pregnancy, and the first three months of life. If so are metabolic abnormalities indicative of impaired carboxylase activities detectable? If so, do the abnormalities resolve with biotin supplementation?
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