Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles

Abstract

Thiamine, a water-soluble vitamin, is essential for normal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety of conditions. To date, however, little is known about the mechanism of thiamine absorption in the native human small intestine. The objective of this study was, therefore, to characterize the mechanism of thiamine transport across the brush-border membrane (BBM) of human small intestine. With the use of purified BBM vesicles (BBMV) isolated from the jejunum of organ donors, thiamine uptake was found to be 1) independent of Na+but markedly stimulated by an outwardly directed H+gradient (pH 5.5in/pH 7.5out); 2) competitively inhibited by the cation transport inhibitor amiloride (inhibitor constant of 0.12 mM); 3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structural analogs (amprolium, oxythiamine, and pyrithiamine), but not by unrelated organic cations (tetraethylammonium, N-methylnicotinamide, or choline); 5) not affected by the addition of ATP to the inside and outside of the BBMV; 6) potential insensitive; and 7) saturable as a function of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 ± 0.08 μM and a maximal velocity of 1.00 ± 0.47 pmol · mg protein−1· 10 s−1. Carrier-mediated thiamine uptake was also found in BBMV of human ileum. These data demonstrate the existence of a Na+-independent, pH-dependent, amiloride-sensitive, electroneutral carrier-mediated mechanism for thiamine absorption in native human small intestinal BBMV.