Identification of unusual oxysterols and bile acids with 7-oxo or 3,5,6-trihydroxy functions in human plasma by charge-tagging mass spectrometry with multistage fragmentation

Abstract

7-Oxocholesterol (7-OC), 5,6-epoxycholesterol (5,6-EC), and its hydrolysis product cholestane-3,5,6-triol (3,5,6-triol) are normally minor oxysterols in human samples; however, in disease, their levels may be greatly elevated. This is the case in plasma from patients suffering from some lysosomal storage disorders, e.g., Niemann-Pick disease type C, or the inborn errors of sterol metabolism, e.g., Smith-Lemli-Opitz syndrome and cerebrotendinous xanthomatosis. A complication in the analysis of 7-OC and 5,6-EC is that they can also be formed ex vivo from cholesterol during sample handling in air, causing confusion with molecules formed in vivo. When formed endogenously, 7-OC, 5,6-EC, and 3,5,6-triol can be converted to bile acids. Here, we describe methodology based on chemical derivatization and LC/MS with multistage fragmentation (MS n ) to identify the necessary intermediates in the conversion of 7-OC to 3-hydroxy-7-oxochol-5-enoic acid and 5,6-EC and 3,5,6-triol to 3,5,6-trihydroxycholanoic acid. Identification of intermediate metabolites is facilitated by their unusual MS n fragmentation patterns. Semiquantitative measurements are possible, but absolute values await the synthesis of isotope-labeled standards.—Griffiths, W. J., I. Gilmore, E. Yutuc, J. Abdel-Khalik, P. J. Crick, T. Hearn, A. Dickson, B. W. Bigger, T. H-Y. Wu, A. Goenka, A. Ghosh, S. A. Jones, and Y. Wang. Identification of unusual oxysterols and bile acids with 7-oxo or 3,5,6-trihydroxy functions in human plasma by charge-tagging mass spectrometry with multistage fragmentation.