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

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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 (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 MS2 ([M]+→) spectra that possessed characteristic neutral loss of 79 Da (pyridine) fragment ions, and MS3 ([M]+  [M−79]+→) spectra that provided additional structurally informative fragment ions.

Introduction

Cerebrotendinous xanthomatosis (CTX; OMIM#213700) is a rare genetic disorder associated with deficient sterol 27-hydroxylase (CYP27A1); an enzyme important in the conversion of cholesterol to cholic and chenodeoxycholic acid (CDCA) (Fig. 1 neutral pathway to primary bile acids). A damaging consequence of CYP27A1 deficiency is the accumulation of 5α-cholestanol (a 5α-dihydro derivative of cholesterol) in the tissues of affected patients. Patients often develop 5α-cholestanol and cholesterol containing xanthomas. Extensive deposition of 5α-cholestanol in the brain [1], is associated with the development of severe neurological dysfunction. In addition to 5α-cholestanol, tissues and blood from untreated CTX patients contain high concentrations of bile acid precursors, in particular 7α-hydroxy-4-cholesten-3-one (C4-7α-ol-3-one, see Fig. 1) [2], [3], [4], [5], [6], [7], [8].

The data reported thus far suggest that a major synthetic pathway to 5α-cholestanol in CTX patients originates from C4-7α-ol-3-one [9], in contrast to a normal synthetic pathway that originates from cholesterol. Both pathways for 5α-cholestanol synthesis appear to converge through 4-cholesten-3-one (C4-3-one, Fig. 1). In vivo studies in CTX patients demonstrated the formation of biliary 5α-cholestanol from intravenous dosed [14C]-labeled C4-3-one [10]. Studies in rabbits [11] and in rats [12] demonstrated that 5α-cholestanol was rapidly found in circulation after oral dosing with [14C]-labeled C4-3-one. Interestingly, prolonged feeding of C4-3-one (0.5%) in birds resulted in severe aortic arteriosclerosis and the accumulation of large amounts of 5α-cholestanol in blood and tissues [12], [13]. The synthesis of 5α-cholestanol from C4-3-one was demonstrated to occur via 5α-cholestan-3-one under in vitro conditions utilizing animal liver tissue preparations [14], [15].

Although in vitro experiments initially suggested C4-3-one was formed from cholesterol in CTX [10], more recent studies indicate a major pathway to C4-3-one in CTX is formation from 4,6-cholestadiene-3-one produced from C4-7α-ol-3-one (Fig. 1) [2], [7]. High concentrations of 4,6-cholestadiene-3-one (500–850 ng/ml) were shown to be present in hydrolyzed serum from untreated CTX patients [2], and the turnover of 4,6-cholestadiene-3-one to C4-3-one and 5α-cholestanol was demonstrated in liver and brain tissue studies [16]. Although C4-7α-ol-3-one can undergo non-enzyme catalyzed dehydration to form 4,6-cholestadiene-3-one, in vitro studies with liver tissue have characterized enzymatic conversion of C4-7α-ol-3-one to 4,6-cholestadiene-3-one and subsequent saturation of the Δ-6 double bond to form C4-3-one [3], [17].

To provide insight into alternate pathways that may be accentuated when bile acid synthesis is perturbed in CTX, we hypothesized a recently described approach utilizing the hybrid Thermo LTQ-Orbitrap instrument coupled with sterol derivatization [18] could be utilized for highly sensitive, selective ESI-MSn analysis of 3-oxo-4-ene sterols present in blood and tissues of CTX patients. The approach involves sterol derivatization with cationic Girard's P (GP) reagent to enhance ESI ionization, as well as to provide MS2 ([M]+→) spectra fragment ions possessing characteristic GP derivative neutral losses of 79 and 107 Da [18], [19] and MS3 ([M]+  [M−79]+→) spectra fragment ions that provide further structural information. The Girard derivatives are resolved using HPLC and are identified by exact mass analysis (±5 ppm), with MS2 and MS3 spectra obtained to confirm sterol identity. Exact mass data is generated in the Orbitrap mass analyzer and MSn spectra are generated in the LTQ linear ion trap (LIT) mass analyzer. We set out to detect sterols possessing a 3-oxo-4-ene or 3-oxo-5(H) saturated structure proposed to be in vivo intermediates in CTX biochemical pathways (Fig. 1) in a targeted manner utilizing derivatization with GP reagent and LC–ESI-MSn analysis with an LTQ-Orbitrap Discovery instrument. We undertook this effort as part of a larger project designed to investigate altered metabolic pathways in CTX, in order to develop improved methods of screening, diagnosis, and treatment for this disorder. We describe here profiling of the free 3-oxo sterols present in plasma from affected patients and from 24-month-old cyp27A1 gene knock out (cyp27A1 −/−) mice deficient in sterol 27-hydroxylase, the enzyme defective in CTX [5], [20], [21]. Bile acid precursors are elevated in these mice, which display a 3- to 5-fold increase in cholesterol 7α-hydroxylase activity [22]. Although large amounts of C4-7α-ol-3-one are present in liver tissue and blood from the mice [5], [20], they do not develop xanthomas, or accumulate 5α-cholestanol to the extent observed in human disease [5], [22], [23]. Recently, significant cerebral accumulation of 5α-cholestanol was noted to occur in 12-month-old cyp27A1 −/− mice, especially in female mice [20], although corresponding to only 3% of the sterol pool versus 20–40% in patients with CTX [24]. The cyp27A1 −/− mice were used to demonstrate formation of cerebral 5α-cholestanol from intravenously dosed [2H]-labeled C4-7α-ol-3-one [20].

To summarize, in this study we report on the utility of GP derivatization and high resolution exact mass LC–ESI-MSn analysis for profiling the free 3-oxo sterols present in plasma from untreated CTX patients and cyp27A1 −/− mice. We previously examined the utility of GP derivatization for quantitative LC–ESI-MS2 analysis of a specific 3-oxo-4-ene sterol elevated in plasma from CTX patients [2], [3], [4], [5], [6], [7], [8]; C4-7α-ol-3-one. We found C4-7α-ol-3-one demonstrated improved utility as a diagnostic marker of disease and to monitor treatment compared to 5α-cholestanol [8]. We anticipate GP-tagged sterols identified with high resolution exact mass LC–ESI-MSn analysis will be amenable to LC–ESI-MS2 analysisperformed with unit resolution instrumentation as previously described for C4-7α-ol-3-one [8], and we describe here preliminary LC–ESI-MS2 experiments to measure 5α-cholestanol precursors We discuss our results in relation to the alternate pathways utilized when bile acid synthesis is perturbed in CTX and in the cyp27A1 −/− mouse.

Section snippets

Chemicals and reagents

4,6-Cholestadiene-3-one, 5α-cholestan-3β-ol (5α-cholestanol), 5α-cholestan-3-one, 4-cholesten-3-one (C4-3-one), cholesterol (5-cholesten-3β-ol), 7α-hydroxy-4-cholesten-3-one (C4-7α-ol-3-one) and 3α,7α,12α-trihydroxy-5β-cholestane were from Steraloids (Newport, RI). 5β-Cholestan-3α-ol (5β-cholestanol) was obtained from Sigma–Aldrich (St Louis, MO). 7α,12α-Dihydroxy-5β-cholestan-3-one was synthesized from 3α,7α,12α-trihydroxy-5β-cholestane using 3α-hydroxysteroid dehydrogenase from Sigma–Aldrich

Derivatization and LC–ESI-MSn methodology

Derivatization of plasma sterols with 10 mM GP reagent in methanol containing 1% acetic acid was performed for 120 min [8], after which time the reaction mixture was injected for automated C18-solid phase extraction (SPE) to remove excess derivatization reagent, with subsequent back-flushing of GP-tagged sterols onto a reversed phase C18-HPLC column for chromatographic separation. Although the removal of plasma cholesterol prior to derivatization with an additional SPE step minimizes generation

Discussion

We present here an example of incorporating a derivatization step for analysis with ESI-MSn, not only to improve analytical sensitivity, but also to tag a specific class of molecules present in a genetic disorder. The selective generation of GP-tagged 3-oxo-4-ene and 3-oxo-5(H) saturated plasma sterols enabled ESI-MSn detection of proposed in vivo sterol intermediates in CTX biochemical pathways (shown in grey, Fig. 1). Sterol detection was accomplished with good sensitivity using conventional

Acknowledgements

This work was supported by an NIH Grant awarded to S.K.E. (DK072187), a Department of Veterans Affairs Merit Award to S.K.E., and a United Leukodystrophy Foundation grant awarded to A.E.D. This work was also supported by a Training grant awarded to A.E.D. from the Sterol and Isoprenoid Diseases (STAIR) consortium. STAIR is a part of NIH Rare Diseases Clinical Research Network (RDCRN). Funding and/or programmatic support for this project has been provided by a grant (1U54HD061939) from NICHD and

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