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The long-term goal of this project is to determine the biotin requirement of normal infants. The specific aims are 1) to accurately measure the total content of biotin and biotin analogs in infant formula, urine, and feces, 2) to determine biotin balance in normal, formula-fed infants fed formula with biotin supplementation at levels similar to human milk and commercial infant formula. Studies in this project are important because several lines of evidence suggest that biotin nutritional status may be inadequate in some normal infants and that the inadequacy could lead to important clinical sequelae that would not be recognized as biotin deficiency. A more sensitive, chemically specific biotin assay based on 125I-avidin and HPLC has been developed that would allow measurement of plasma biotin and urinary excretion of biotin and biotin analogs as well as a more accurate estimate of free techniques, biotin balance will be determined before 30 and after 60 days of age in normal infants fed an infant formula. Using these same assays, biotin nutritional status will be determined longitudinally from 7 to 112 days of age in infants fed formula supplemented to reach three different total biotin concentrations: 1) a concentration equal to the first quartile of the distribution of biotin concentrations in human milk, 2) a second concentration equal to the third quartile of the same distribution, and 3) a third concentration typical of commercial infant formula. In these longitudinal studies, biotin depletion at the tissue level will also be assessed in two ways: 1) Deficient activity of the biotin-dependent enzyme 3-methylcrotonyl-CoA carboxylase impairs metabolism of leucine and leads to abnormal excretion of 3-hydroxyisovaleric acid in the urine. Gas chromatography/mass spectrometry with deuterated 30HIV as internal standard will be used to quantitate 3-HIV excretion. 2) Deficient activity of the biotin-dependent enzyme propionyl-CoA carboxylase causes an increase in the percentage composition of odd chain saturated fatty acid in the phospholipid fraction of plasma. Fatty acid composition in plasma phospholipid will be measured by thin layer chromatography/gas chromatography.

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