In this study we profile free 3-oxo
sterols present in plasma from patients affected with the
neurodegenerative disorder of
sterol and
bile acid metabolism
cerebrotendinous xanthomatosis (CTX), utilizing a combination of charge-tagging and LC-ESI-MS(n) performed with an LTQ-Orbitrap Discovery instrument. In addition, we profile
sterols in plasma from 24-month-old cyp27A1 gene knockout mice lacking the
enzyme defective in CTX. Charge-tagging was accomplished by reaction with cationic Girard's P (GP)
reagent 1-(carboxymethyl) pyridinium
chloride hydrazide, an approach uniquely suited to studying the 3-oxo
sterols that accumulate in CTX, as Girard's
reagent reacts with the
sterol oxo moiety to form charged
hydrazone derivatives. The ability to selectively generate GP-tagged 3-oxo-4-ene and 3-oxo-5(H) saturated plasma
sterols enabled ESI-MS(n) analysis of these
sterols in the presence of a large excess (3 orders of magnitude) of
cholesterol. Often
cholesterol detected in
biological samples makes it challenging to quantify minor
sterols, with
cholesterol frequently removed prior to analysis. We derivatized plasma (10 μl) without SPE removal of
cholesterol to ensure detection of all
sterols present in plasma. We were able to measure
4-cholesten-3-one in plasma from untreated CTX patients (1207±302 ng/ml, mean±SD, n=4), as well as other intermediates in a proposed pathway to 5α-cholestanol. In addition, a number of
bile acid precursors were identified in plasma using this technique. GP-tagged
sterols were identified utilizing high resolution exact mass spectra (±5 ppm), as well as MS(2) ([M](+)→) spectra that possessed characteristic neutral loss of 79Da (
pyridine) fragment
ions, and MS(3) ([M](+)→[M-79](+)→) spectra that provided additional structurally informative fragment
ions.