Uptake of riboflavin by isolated rat liver cells

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/(10(6) cells X 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 Km of 12 +/- 1.3 microM and Vmax of 82.3 +/- 9.1 pmol/(10(6) cells X minute) were obtained for control and riboflavin-sufficient rats while a similar Km but higher Vmax were obtained with deficient animals. Correspondence of the Km 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.