Characterisation of non-transferrin-bound iron (ferric citrate) uptake by rat hepatocytes in culture

Abstract

Under conditions of iron overload plasma transferrin can be fully saturated and the plasma can transport non-transferrin-bound Fe which is rapidly cleared by the liver. Much of this Fe is complexed by citrate. The aim of the present work was to characterise the mechanisms by which Fe-citrate is taken up by hepatocytes using a rat hepatocyte cell culture model. The cells, after one day in culture, were incubated with 59 Fe -labelled Fe-citrate for varying time periods, then washed and Fe uptake to the membrane and intracellular compartments of the cell was determined by radioactivity measurements. Maximal rates of internalisation of Fe occurred at a Fe:citrate molar ratio of 1:100 or greater, a pH of approximately 7.4 and an extracellular Ca 2+ concentration of 1.0 mM. Fe uptake showed Michaelis–Menten kinetics and was a temperature-dependent process. The K m and V max for Fe internalisation by the cells at 37°C were approximately 7 μM and 2 nmol/mg DNA/min (25×10 6 atoms/cell/min), respectively; and the Arrhenius activation energy was 35 kJ/mol. The transition metals, Zn 2+, Co 2+ and Ni 2+, inhibited Fe uptake when used at 10 and 100 times the concentration of Fe. The rate of Fe internalisation from Fe-citrate was found to be approximately 20 times as great as that from Fe-transferrin with Fe concentrations of 1 and 2.5 μM for both forms of Fe. The rate of Fe uptake by iron-loaded hepatocytes obtained from rats which had been fed carbonyl Fe was not significantly different from that by normal hepatocytes. These experiments show that rat hepatocytes in primary culture have a high capacity to take up non-transferrin-bound Fe in the form of Fe-citrate and that uptake occurs by facilitated diffusion. The iron transport process does not appear to be regulated by cellular Fe levels.