Thiamin transport by human erythrocytes and ghosts

Abstract

Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20 degrees C in the presence of [3H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na+ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 microM and at 37 degrees C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes, Km = 0.12, 0.11 and 0.10 microM and Jmax = 0.01, 0.02 and 0.03 pmol.microliter-1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts, Km = 0.16 and 0.51 microM and Jmax = 0.01 and 0.04 pmol.microliter-1 intracellular water after 15 and 30 min incubation times, respectively. At 20 degrees C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence of D-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein- (probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN- showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.

Thiamine transport by erythrocytes and ghosts in thiamine‐responsive megaloblastic anaemia

“…Since thiamine transport is basically similar in plasma membranes from different cells (Casirola et al, 1990), erythrocytes were selected for our study according to their availability.…”

“…Materials: Two isotopically labelled thiamines, prepared by Amersham, International, plc, Amersham, UK, were used: for uptake experiments, tritiated thiamine (EaH]T) (specific activity 7.5 Ci/mmol, 95% purity as evaluated chromatographically by Casirola et al, 1990); for TPKase assay, thiazole-[2-14C]thiamine hydrochloride (I-14C]T) (specific activity 14-25 mCi/mmol). S-benzoyloxymethyl (BOM)-thiamine was kindly supplied by Bioindustria, Novi Ligure, Italy.…”

“…Erythrocyte and ghost preparation: Erythrocytes were prepared according to Casirola et al (1990) 24-48 h after collection, from human blood samples stored at 4°C in citrate buffer (pH 7.4) containing glucose and adenine. Right-side-out, pink ghosts resealed to contain 145mmol/L NaC1 were prepared from the above-mentioned erythrocytes, immediately before use, following the procedure of Schwoch and Passow (1973) as described by Casirola et al (1990).…”

“…Erythrocytes and ghosts from patients exhibited no saturable component, this abnormality being specific for the patients and not shared by their parents. It is concluded that the cells from thiamine-responsive megaloblastic anaemia patients contain low levels of thiamine compounds, probably due to their inability to take up and retain physiological concentrations of thiamine, as a result of the lack of the saturable, specific component of transport and reduced thiamine pyrophosphokinase.The kinetic properties of thiamine transport by human erythrocytes and ghosts in vitro have been defined recently (Casirola et al, 1990). In both preparations, thiamine uptake at physiological concentrations is a specific process, independent of the presence of Na ÷, unaffected by changes in membrane potential and predominantly accounted for by a saturable mechanism.…”

Cloning of the Human Thiamine Transporter, a Member of the Folate Transporter Family

“…Consistent with such an uptake mechanism, the thiamine uptake into placental brush border membrane vesicles was abolished in the presence of proton ionophores and unaffected by the presence of an inside-negative membrane potential. Existence of a similar uptake mechanism for thiamine has also been described in intestine (8) and erythrocytes (10).…”