Tandem mass spectrometric determination of atypical 3β-hydroxy-Δ5-bile acids in patients with 3β-hydroxy-Δ5-C27-steroid oxidoreductase deficiency: Application to diagnosis and monitoring of bile acid therapeutic response

Abstract

BACKGROUND: 3β-Hydroxy-Δ5-C27-steroid oxidoreductase (HSD3B7) deficiency, a progressive cholestatic liver disease, is the most common genetic defect in bile acid synthesis. Early diagnosis is important because patients respond to oral primary bile acid therapy, which targets the negative feedback regulation for bile acid synthesis to reduce the production of hepatotoxic 3β-hydroxy-Δ5-bile acids. These atypical bile acids are highly labile and difficult to accurately measure, yet a method for accurate determination of 3β-hydroxy-Δ5-bile acid sulfates is critical for dose titration and monitoring response to therapy. METHODS: We describe a electrospray ionization LC-MS/MS method for the direct measurement of atypical 3β-hydroxy-Δ5-bile acid sulfates in urine from patients with HSD3B7 deficiency that overcomes the deficiencies of previously used GC-MS methods. RESULTS: Separation of sulfated 3β-hydroxy-Δ5-bile acids was achieved by reversed-phase HPLC in a 12-min analytical run. The mean (SE) urinary concentration of the total 3β-sulfated-Δ5-cholenoic acids in patients with HSD3B7 deficiency was 4650 (1711) μmol/L, approximately 1000-fold higher than in noncholestatic and cholestatic patients with intact primary bile acid synthesis. GC-MS was not reliable for measuring 3β-hydroxy-Δ5-bile acid sulfates; however, direct analysis of urine by fast atom bombardment mass spectrometry yielded CONCLUSIONS: The tandem mass spectrometry method described here for the measurement of 3β-hydroxy-Δ5-bile acid sulfates in urine can be applied to the diagnosis and accurate monitoring of responses to primary bile acid therapy in HSD3B7 patients.