Determination of the glucurono-conjugated position in bile alcohol glucuronides present in a patient with cerebrotendinous xanthomatosis

Abstract

The determination of the glucurono-conjugated position in three bile alcohol glucuronides secreted in bile of a patient with cerebrotendinous xanthomatosis was carried out 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 5β-cholestane-3α,7α,12α,25-tetrol, 5β-cholestane-3α,7α,12α,23-tetrol, and 5β-cholestane-3α,7α,12α,23,25-pentol. Proton and 13C nuclear magnetic resonance spectra of the two biliary bile alcohol glucuronides, 5β-cholestane-3α,7α,12α,25-tetrol glucuronide and 5β-cholestane-3α,7α,12α,23,25-pentol glucuronide were identical with those of the synthetic glucuronide 7α,12α,25-trihydroxy-5β-cholestane-3α-Oβ- d-glucopyranosyluronic acid and the isolated glucuronide 3α,7α,12α,25-tetrahydroxy-5β-cholestane-23-O-β- 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 13C chemical shift data with that of the unconjugated bile alcohol, 5β-cholestane-3α,7α,12α,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 3α,7α,12α-trihydroxy-5β-cholestane-23-O-β- d-glucopyranosyluronic acid.