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 13C chemical shift data with that of the unconjugated
pentol, 5 beta-cholestane-3 alpha, 7 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-cholestane-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.