Examination of the Sephadex G10 beads uptake method for determination of bile salt monomer concentration in...

Experiments have been undertaken to examine the validity of the method of Ammon and Walter for the determination of bile acid monomer concentrations in taurocholate-lecithin (TC-L) solutions. The procedure involves, basically, the uptake of the bile acid monomer by Sephadex G10 beads (exclusion limits 700 Da) in the presence of a suitable aqueous phase marker (raffinose). Several of the experimental variables involved in the bead-washing technique have been critically studied, and good precision in data (SD in the range of +/- 2-5% of the mean value) has been obtained. Monomer concentrations determined in the absence of lecithin over a wide range (10-70 mM) of TC were found to be in agreement with those obtained by Ammon. Most important, however, is the finding that TC monomer concentrations at constant NMBS were found to be constant in all solutions containing or not containing lecithin [here, the nonmixed micelle bile salt (NMBS) is the total bile salt concentration, but not including species involving lecithin, and is determined by dialysis equilibrium experiments].