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

Abstract

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 5β-cholestane-3α, 7α, 12α, 25-tetrol and 5β-chole-stane-3α, 7α, 12α, 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, 7α, 12α, 25-trihydroxy-5β-cholestane-3α-O-β-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, 5β-cholestane-3α, 7α, 12α, 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 3α, 7α, 12α, 25-tetrahydroxy-5β-cholestane-23-O-β-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.