Manganese cell labeling of murine hepatocytes using manganese(III)‐transferrin

Abstract

Manganese(III)-transferrin [Mn(III)-Tf] was investigated as a way to accomplish manganese-labeling of murine hepatocytes for MRI contrast. It is postulated that Mn(III)-Tf can exploit the same transferrin-receptor-dependent and -independent metabolic pathways used by hepatocytes to transport the iron analog Fe(III)-Tf. More specifically, it was investigated whether manganese delivered by transferrin could give MRI contrast in hepatocytes. Comparison of the T1 and T2 relaxation times of Mn(III)-Tf and Fe(III)-Tf over the same concentration range showed that the r1 relaxivities of the two metalloproteins are the same in vitro, with little contribution from paramagnetic enhancement. The degree of manganese cell labeling following incubation for 2-7 h in 31.5 microm Mn(III)-Tf was comparable to that of hepatocytes incubated in 500 microm Mn2+ for 1 h. The intrinsic manganese tissue relaxivity between Mn(III)-Tf-labeled and Mn2+-labeled cells was found to be the same, consistent with Mn(III) being released from transferrin and reduced to Mn2+. For both treatment regimens, manganese uptake by hepatocytes appeared to saturate in the first 1-2 h of the incubation period and may explain why the efficiency of hepatocyte cell labeling by the two methods appeared to be comparable in spite of the approximately 16-fold difference in effective manganese concentration. Hepatocytes continuously released manganese, as detected by MRI, and this was the same for both Mn2+- and Mn(III)-Tf-labeled cells. Manganese release may be the result of normal hepatocyte function, much in the same way that hepatocytes excrete manganese into the bile in vivo. This approach exploits a biological process-namely receptor binding, endocytosis and endosomal acidification-to initiate the release of an MRI contrast agent, potentially conferring more specificity to the labeling process. The ubiquitous expression of transferrin receptors by eukaryotic cells should make Mn(III)-Tf particularly useful for manganese labeling of a wide variety of cells both in culture and in vivo.