Search

Purpose of Review: To update researchers of recently discovered metabolites of cholesterol and of its precursors and to suggest relevant metabolic pathways., Recent Findings: Patients suffering from inborn errors of sterol biosynthesis, transport and metabolism display unusual metabolic pathways, which may be major routes in the diseased state but minor in the healthy individual. Although quantitatively minor, these pathways may still be important in healthy individuals. Four inborn errors of metabolism, Smith-Lemli-Opitz syndrome, cerebrotendinous xanthomatosis and Niemann Pick disease types B (NPB) and C (NPC) result from mutations in different genes but can generate elevated levels of the same sterol metabolite, 7-oxocholesterol, in plasma. How this molecule is metabolized further is of great interest as its metabolites may have an important role in embryonic development. A second metabolite, abundant in NPC and NPB diseases, cholestane-3β,5α,6β-triol (3β,5α,6β-triol), has recently been shown to be metabolized to the corresponding bile acid, 3β,5α,6β-trihydroxycholanoic acid, providing a diagnostic marker in plasma. The origin of cholestane-3β,5α,6β-triol is likely to be 3β-hydroxycholestan-5,6-epoxide, which can alternatively be metabolized to the tumour suppressor dendrogenin A (DDA). In breast tumours, DDA levels are found to be decreased compared with normal tissues linking sterol metabolism to cancer., Summary: Unusual sterol metabolites and pathways may not only provide markers of disease, but also clues towards cause and treatment.

The application of Gas Chromatography (GC)-Atmospheric Pressure Chemical Ionization (APCI)-Time-of-Flight Mass Spectrometry (TOF-MS) is presented for sterol analysis in human plasma. A commercial APCI interface was modified to ensure a well-defined humidity which is essential for controlled ionization. In the first step, optimization regarding flow rates of auxiliary gases was performed by using a mixture of model analytes. Secondly, the qualitative and quantitative analysis of sterols including oxysterols, cholesterol precursors, and plant sterols as trimethylsilyl-derivatives was successfully carried out. The characteristics of APCI together with the very good mass accuracy of TOF-MS data enable the reliable identification of relevant sterols in complex matrices. Linear calibration lines and plausible results for healthy volunteers and patients could be obtained whereas all mass signals were extracted with an extraction width of 20 ppm from the full mass data set. One advantage of high mass accuracy can be seen in the fact that from one recorded run any search for m/z can be performed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.