Background: To quantify transmembrane transport of dipeptides by PepT1, passive uptake (non-PepT1 mediated) must be subtracted from total (measured) uptake. Three methods have been described to estimate passive uptake: perform experiments at cold temperatures, inhibit target dipeptide uptake with a greater concentration of a second dipeptide, or use modified Michaelis-Menten kinetics. We hypothesized that performing uptake experiments at pH 8.0 would estimate passive uptake accurately, because PepT1 requires a proton gradient. Our aim was to determine the most accurate method to estimate passive uptake. Methods: Caco-2 cells were incubated with various concentrations of glycyl-sarcosine (gly-sar) at pH 6.0 and at 37°C to measure total uptake. Passive uptake was estimated: (1) by incubating Caco-2 cells with varying concentrations of gly-sar at 4°C, (2) in the presence of 50 mM glycyl-leucine, (3) in solution at pH 8.0, or (4) using modified Michaelis-Menten kinetics. PepT1-mediated uptake was calculated by subtracting passive uptake from total uptake. Km, Vmax, and % gly-sar transported by PepT1 were calculated and compared. Results: Km, Vmax, and % gly-sar transported by PepT1 varied from 0.7 to 2.4 mM, 8.4 to 21.0 nmol/mg protein/10 min, and 69% to 87%, respectively. Uptakes calculated with cold, 50 mM gly-leu and using modified Michaelis-Menten kinetics were similar but differed significantly from uptake at pH 8.0 (P < 0.001). Conclusions: Estimating passive uptake at pH 8.0 does not appear to be accurate. Measuring uptake at cold temperatures or in the presence of a greater concentration of a second dipeptide, and confirming results with modified Michaelis-Menten kinetics is recommended.
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