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THIAMINE UPTAKE BY HUMAN FIBROBLASTS

Thiamine requiring enzymes are commonly studied in cultured fibroblasts but the mechanism of thiamine accumulation had not been defined in these cells. Thiamine is concentrated greater than in the medium by 30-60 minutes and reaches apparent concentrations 6 times the medium by 2-3 hours. Kinetic studies demonstrate that uptake is saturable and the apparent Km(.15-3 μM) and Vmax (.6-.8 p moles/mg/min) are similar to those in gut and brain. However, the uptake process is quite different. Accumulation is highly specific but not energy dependent. Before thiamine is phosphorylated it is bound to at least two <40,000 MW cytoplasmic proteins. Uptake and binding are competitively inhibited by pyrithiamin but only slightly by TP or TPP. Thiamine efflux is biphasic with rapid efflux occuring over 30-60 minutes followed by a slow phase lasting at least several hours, probably reflecting phosphorylation.We conclude that thiamine uptake in these cells is more comparable to steroid hormone uptake than to thiamine uptake by specialized transport tissue.