Profiling sterols in cerebrotendinous xanthomatosis: Utility of Girard derivatization and high resolution exact mass LC-ESI-MSn analysis

Andrea De Barber, Yana Sandlers, Anuradha Pappu, Louise S. Merkens, Paul Duell, Steven R. Lear, Sandra K. Erickson, Robert D. Steiner

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

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-MSn 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-MSn 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 (10μl) 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±302ng/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 (±5ppm), as well as MS2 ([M]+→) spectra that possessed characteristic neutral loss of 79Da (pyridine) fragment ions, and MS3 ([M]+→[M-79]+→) spectra that provided additional structurally informative fragment ions.

Original languageEnglish (US)
Pages (from-to)1384-1392
Number of pages9
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume879
Issue number17-18
DOIs
StatePublished - May 15 2011

Fingerprint

Cerebrotendinous Xanthomatosis
Sterols
Plasmas
Cholesterol
Bile Acids and Salts
Cholestanol
Ions
Hydrazones
Gene Knockout Techniques
Metabolism
Knockout Mice
Neurodegenerative Diseases
Chlorides

Keywords

  • Bile alcohols
  • Derivatization
  • FTMS
  • Genetic disorders
  • Keto sterols
  • LC-ESI-MSn
  • Multi-stage CID fragmentation

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Cell Biology
  • Clinical Biochemistry

Cite this

Profiling sterols in cerebrotendinous xanthomatosis : Utility of Girard derivatization and high resolution exact mass LC-ESI-MSn analysis. / De Barber, Andrea; Sandlers, Yana; Pappu, Anuradha; Merkens, Louise S.; Duell, Paul; Lear, Steven R.; Erickson, Sandra K.; Steiner, Robert D.

In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol. 879, No. 17-18, 15.05.2011, p. 1384-1392.

Research output: Contribution to journalArticle

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