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 alcohol glucuronides secreted in bile of a patient with cerebrotendinous 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 biliary bile alcohol glucuronides, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol glucuronide and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol glucuronide were identical with those of the synthetic glucuronide 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-glucopyranosyluronic acid from urine of a patient with cerebrotendinous xanthomatosis, respectively. Hence, the glucurono-conjugated positions of the biliary 25-tetrol glucuronide and the biliary 23,25-pentol glucuronide were C-3 and C-23, respectively. By comparison of the C-13 chemical shift data with that of the unconjugated bile alcohol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23-tetrol, the glucurono-conjugated position of the natural 23-tetrol glucuronide was determined to be C-23. Thus, the natural 23-tetrol glucuronide can be formulated as 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestane-23-O-beta-D-glucopyranosyluronic acid.