DETERMINATION OF THE GLUCURONO-CONJUGATED POSITION IN BILE ALCOHOL GLUCURONIDES PRESENT IN A PATIENT WITH CEREBROTENDINOUS XANTHOMATOSIS

The determination of the glucurono-conjugated position in three bile a lcohol glucuronides secreted in bile of a patient with cerebrotendinou s xanthomatosis was carried our by a nuclear magnetic resonance study. The bile sample was extracted with ethanol and chromatographed on an ion-exchange column, a reverse-phase partition column and a silica gel column to isolate glucurono-conjugates of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 23-tetrol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol . Proton and C-13 nuclear magnetic resonance spectra of the two biliar y bile alcohol glucuronides, 5 beta-cholestane-3 alpha,7 alpha,12 alph a,25-tetrol glucuronide and 5 beta-cholestane-3 alpha,7 alpha,12 alpha ,23,25-pentol glucuronide were identical with those of the synthetic g lucuronide 7 alpha,12 alpha,25-trihydroxy-5 beta-cholestane-3 alpha-O beta-D-glucopyranosyluronic acid and the isolated glucuronide 3 alpha, 7 alpha,12 alpha,25-tetrahydroxy-5 beta-cholestane-23-O-beta-D-glucopy ranosyluronic acid from urine of a patient with cerebrotendinous xanth omatosis, respectively. Hence, the glucurono-conjugated positions of t he biliary 25-tetrol glucuronide and the biliary 23,25-pentol glucuron ide were C-3 and C-23, respectively. By comparison of the C-13 chemica l shift data with that of the unconjugated bile alcohol, 5 beta-choles tane-3 alpha,7 alpha,12 alpha,23-tetrol, the glucurono-conjugated posi tion of the natural 23-tetrol glucuronide was determined to be C-23. T hus, the natural 23-tetrol glucuronide can be formulated as 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane-23-O-beta-D-glucopyranosyl uronic acid.