Abstract
Some commercial preparations of common natural conjugated bile salts contain impurities (e.g., amines, lipids, and calcium) that are likely to affect their physicochemical properties. A method was developed for purifying commercial preparations of sodium salts of glycine- and taurine-conjugated bile acids. The method consists of passage of a dilute aqueous solution of the sodium bile salt through three columns in sequence: graphitized carbon, a hydrophobic bonded octadecylsilane (C18) cartridge, and a calcium-chelating resin. The final solution was extracted with chloroform, and the purified bile salt was then isolated by freeze-drying, with a yield of 65-75%. Each bile salt purified by this method was compared with the corresponding bile salt purified by conventional adsorption chromatography on a silicic acid column, using a mixture of methanol and chloroform as eluant. Purity was assessed by visible spectra, by surface tension measurements (using the maximum bubble-pressure method and a Wilhelmy wire method), by chloroform extractability of impurities in the conjugated bile acid, by liposome solubilization, and by chemical analysis of the calcium content. Both purification methods removed colored and surface-active impurities, but the new method was always as or more effective than silicic acid column chromatography. Calcium ion, present in commercial bile salts in concentrations up to 16 mmol/mol bile salt, was removed completely by the three-column method, but not by silicic acid chromatography. The new method is thus a simple, rapid, and efficient procedure for purification of the sodium salts of glycine- and taurine-conjugated bile acids for physicochemical measurements, in which elimination of surface-active impurities and polyvalent cations is desired.
Highlights
Some commercial preparations of common natural conjugated bile salts contain impurities that are likely to affect their physicochemical properties
In vivo, mixed micelles containing conjugated bile salt anions and phosphatidylcholine are present in bile [6] and mixed micelles containing monoacylglycerol and fatty acids are found in intestinal content during fat digestion [7].Bile salt anions, like detergent anions, bind counterions, [8,9,10,11], a process that may enhance the absorption of divalent cations such as calcium [12] and iron [12, 13]
For C-Gly and CDC-Gly, the commercial samples were more surface-activethan samples purified by the new method, showing lower surface tensionsboth below and abovethe “critical micellization concentration (CMC)”(Fig. 1, upper panels)
Summary
Some commercial preparations of common natural conjugated bile salts contain impurities (e.g., amines, lipids, and calcium) that are likely to affect their physicochemical properties. Purity was assessed by visible spectra, by surface tension measurements (using the maximum bubblepressure method and a Wilhelmy wire method), by chloroform extractability of impurities in the conjugated bile acid, by liposome solubilization, and by chemical analysis of the calcium content. Both purification methods removed colored and surface-activeimpurities,but the new method was always as or more effective than silicic acid column chromatography. Method for removal of surface-active impurities and calcium from conjugated bile salt preparations: comparison with silicic acid chromatography. Spiriti in Saxia, Lungotevere in Sassia, 1,1-00193Roma,Italy. 90whomcorrespondence shouldbe addressedat present address: Liver/Gastrointestinal Department, Room C-2-111, Academic
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