Abstract
Using thin-layer chromatography, bile alcohol glucuronides were found with taurine- and glycine-conjugated bile acids in the bile of four patients with cerebrotendinous xanthomatosis. The concentration of the bile alcohol glucuronides was 1.7-5.2 times higher than that of the conjugated bile acids. Detectable amounts of unconjugated bile alcohols were not found in the bile of these patients. The bile alcohol glucuronides were isolated from the bile of one of the patients by means of preparative thin-layer chromatography. Treatment with beta-glucuronidase of the bile alcohol glucuronides liberated glucuronic acid and a mixture of bile alcohols. More than 90% of the liberated bile alcohols was 5 beta-cholestane-3 alpha, 7 alpha, 25-tetrol, and lesser amounts of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23-tetrol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24-tetrol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23, 25-pentol, and 5 beta-cholestane-3 alpha,-7 alpha, 12 alpha, 24 alpha, 25-pentol were also obtained. The bile alcohol glucuronides were not oxidized by the treatment with 3 alpha-hydroxysteroid dehydrogenase, indicating that the glucuronide moiety was at 3 alpha-hydroxyl position of the bile alcohols. Comparison of the mass spectra of the acetylated and methylated derivatives of the natural glucuronides and the synthetic 7 alpha, 12 alpha, 25-triacetoxy-5 beta-cholestan-3 alpha-O-(methyl 2,3,4-tri-O-acetyl-beta-D-glucopyranosyluronate) also indicated that the bile alcohol glucuronides consisted of mainly 5 beta - cholestane - 3 alpha, 7 alpha, 12 alpha, 25 - tetrol - glucuronide.
Highlights
Using thin-layer chromatography, bile alcohol glucuronides were found with taurine- and glycineconjugated bile acids in the bile of four patients with cerebrotendinous xanthomatosis
I n this report we describe the occurrence of bile alcohol glucuronides as major constituents in the bile of four patients \vith cerebrotendinous xanthomatosis (CTX)
Bile salt composition was determined by thin-layer chromatography (TLC)-direct densitometry (10)
Summary
Melting points were determined with a Kofler-hot stage apparatus, and are uncorrected. After development in chloroform-methanol-acetic acid-water 13:4:2: 1 (by vol), bile salt bands were detected by exposure of the dried plates to iodine vapor. For determination o f biliary bile salt composition, TLC-direct densitometry was carried out according to the procedures described previously ( 10). A portion of the bile sample from patient 1 was diluted Ivith 50 in[1] of water containing 1 rnl of concentrated NH,OH, and \vas extracted with four 50-ml portions of ethyl acetate. T h e residue was examined 11y TLC using solvent systems ethyl acetate-acetone 7:3 (by vol) a n d benzene-isopropanol-acetic acid 30: I O : 1 (by vol) and by GLC-MS using 3% O V - 17 column for the detection of unconjugated bile alcohols. T h e ethyl acetate extracts were combined, washed with water, dried over anhydrous Na2S04,and evaporated to dryness. IR vmaXKHr(cmp1')7:50, 1725, 1240, and 1030 (methyl ester and acetate).NMR(S ppm):0.76 (s, 3H, 18-CH,), 0.86 (s, 3 H , 19-CH3),0.98 (d,J=6 Hz, 3 H , Zl-CH,), 1.30 (s, 6 H , 26--CH3 and 27-CH3), 1.98 (s, 3 H , -O
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