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Article

Riboflavin Phosphorylation Is the Crucial Event in Riboflavin Transport by Isolated Rat Enterocytes¹

Giulia Gastaldi, Giuseppina Ferrari, Anna Verri, Donatella Casirola^*, Maria Novella Orsenigo and Umberto Laforenza²

Institute of Human Physiology, University of Pavia, 27100 Pavia, Italy; ^* Department of Pharmacology and Physiology, UMD, New Jersey Medical School, Newark, NJ 07103; and Department of General Physiology and Biochemistry, University of Milan, 23100 Milan, Italy

²To whom correspondence and reprint requests should be addressed.

In isolated rat enterocytes, both normoenergized (normal) and de-energized with rotenone, riboflavin intracellular metabolic processes, operating in association with a membrane-specific transport mechanism, were investigated. The contents of unlabeled (endogenous) and labeled (exogenous) flavins [riboflavin (RF), flavin mononucleotide (FMN), flavin adenindinucleotide (FAD)] were determined by HPLC before and after incubation with tritiated RF . In normoenergized enterocytes, total labeled RF content (i.e., total uptake, the sum of RF membrane transport and intracellular metabolism) increased steadily to a plateau after 20 min incubation; FMN and FAD contents reached a plateau between 3 and 20 min, whereas free RF content increased constantly. The phosphorylated forms prevailed over the free form (60% of total flavins). In de-energized enterocytes, RF total uptake was significantly lower than in normoenergized enterocytes and reached a plateau after only 3 min incubation. FMN and FAD contents were significantly lower than in normoenergized enterocytes, and free RF represented the prevailing form of flavins (70% of total RF ). In both normoenergized and de-energized enterocytes, the contents of unlabeled total RF, FMN and FAD decreased significantly after 20 min incubation, whereas free RF increased significantly only in normoenergized enterocytes. After 20 min incubation, the RF structural analog 8-dimethyl-amino-8-demethyl-RF caused a significant decrease of all flavin contents, whereas 5'-deoxy-RF decreased only the total and free RF contents. Results directly confirmed the leading role of metabolic processes such as phosphorylation in RF transport by isolated small intestinal enterocytes.

KEY WORDS: • riboflavin • isolated enterocytes • intestinal absorption • riboflavin metabolism • rats

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