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

摘要

7-Oxocholesterol (7-OC), 5,6-epoxycholesterol (5,6-EC), and its hydrolysis product cholestane-3 beta,5 alpha,6 beta-triol (3 beta,5 alpha,6 beta-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 (MSn) 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 beta,5 alpha,6 beta-triol to 3 beta,5 alpha,6 beta-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 beta,5 alpha,6 beta triol to 3 beta,5 alpha,6 beta-trihydroxycholanoic acid. Identification of intermediate metabolites is facilitated by their unusual MSn fragmentation patterns. Semiquantitative measurements are possible, but absolute values await the synthesis of isotopelabeled standards.-Griffiths,