DETERMINATION OF THE GLUCURONO-CONJUGATED POSITION IN BILE ALCOHOL GLUCURONIDES EXCRETED IN URINE OF A PATIENT WITH CEREBROTENDINOUS XANTHOMATOSIS BY A NUCLEAR-MAGNETIC-RESONANCE STUDY

This paper describes the determination of the glucurono-conjugated position in two bile alcohol glucuronides excreted in urine of a patient with cerebrotendinous xanthomatosis by a nuclear magnetic resonance study. The urine sample was extracted with reversed-phase resin, and chromatographed on a reversed-phase partition column and a silica gel column to isolate glucurono-conjugates of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol. Proton and carbon-13 nuclear magnetic resonance spectra of the natural tetrol glucuronide were identical with those of the chemically synthesized tetrol glucuronide, 7 alpha,12 alpha,25-trihydroxy-5 beta-cholestane-3 alpha-O-beta-D-glucopyranosyluronic acid. Hence, the glucurono-conjugated position of the natural tetrol glucuronide was determined to be the C-3 position. By comparison of the C-13 chemical shift data with that of the unconjugated pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol, the glucurono-conjugated position of the natural pentol glucuronide was determined to be C-23. Thus the natural pentol glucuronide can be formulated as 3 alpha,7 alpha,12 alpha,25-tetrahydroxy-5 beta-cholestan-23-O-beta-D-glucopyranosyluronic acid. The difference in the glucurono-conjugated position between the 25-tetrol glucuronide and the 23,25-pentol glucuronide indicates that the former is not the biosynthetic precursor of the latter.