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Uptake of Riboflavin by Isolated Rat Liver Cells¹

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322

The effect of riboflavin status on uptake was investigated in hepatocytes isolated from control, riboflavin-sufficient and riboflavin-deficient rats. The uptake exhibited biphasic characteristics with an initial rapid phase [13.2 ± 1.8 pmol/(10⁶ cells·minute)] for the first couple of minutes followed by a second slower phase which continued for over an hour. The accumulation of riboflavin at near equilibrium conditions was 2.5- and 5.2-fold greater than external concentration in control and riboflavin-deficient cells, respectively. An apparent K_m of 12 ± 1.3 µM and _max of 82.3 ± 9.1 pmol/(10⁶ cells·minute) were obtained for control and riboflavin-sufficient rats while a similar K_m but higher _max were obtained with deficient animals. Correspondence of the K_m to that of flavokinase for riboflavin suggested the possibility that uptake of the vitamin may occur via metabolic trapping, i.e., phosphorylation. As substantiation of this, the rate of uptake was decreased by lumiflavin and 2'-hydroxyethylflavin, which are competitive inhibitors, and by 7,8-dichloroflavin, a substrate for flavokinase. Furthermore, the uptake was found to be temperature-dependent and studies with carbonylcyanide-p-trifluoro-methoxyphenylhydrazone (FCCP) and ethionine indicated a requirement for ATP. These results showed that overall, entry of riboflavin into hepatocytes occurs predominantly by a facilitated diffusion process followed by rapid trapping by flavokinase-catalyzed phosphorylation to FMN.

KEY WORDS: • riboflavin uptake • rat hepatocytes • metabolic trapping

¹ Supported in part by U.S. Public Health Service, National Institutes of Health grant AM26746.

² To whom reprint requests should be sent.

Manuscript received 13 January 1983.