Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry.

Jonas Abdel-Khalik,William J Griffiths,Peter J Crick,Yuqin Wang,Eylan Yutuc

doi:10.3390/molecules24030597

Abstract

Enzyme-assisted derivatization for sterol analysis (EADSA) is a technology designed to enhance sensitivity and specificity for sterol analysis using electrospray ionization–mass spectrometry. To date it has only been exploited on sterols with a 3β-hydroxy-5-ene or 3β-hydroxy-5α-hydrogen structure, using bacterial cholesterol oxidase enzyme to convert the 3β-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3α-hydroxysteroid dehydrogenase (3α-HSD) for cholesterol oxidase, making the method applicable to sterols with a 3α-hydroxy-5β-hydrogen structure. The 3α-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C27 acids give an intense molecular ion ([M]+) upon electrospray ionization and informative fragmentation spectra. The method shows promise for analysis of bile alcohols and 3α-hydroxy-5β-C27-acids, enhancing the range of sterols that can be analyzed at high sensitivity in sterolomic studies.

Highlights

Sterols represent one of the major classes of lipids found in living systems [1]
For decades there was little interest in these intermediates, in recent years the situation has changed with the realization that intermediates in bile acid biosynthesis are ligands to nuclear receptors, including the liver X receptors (LXRs, NR1H3, NR1H2) [10,11,12,13], farnesoid X receptor (FXR, NR1H4) [14], pregnane X receptor (PXR, known as xenobiotic sensing nuclear receptor, SXR, NR1I2) [15,16], RAR-related orphan receptor γt (RORγt, NR1F3) [17], and estrogen receptors (ERs, NR3A1, NR3A2) [18]
Cholesterol metabolites have traditionally been analyzed by gas-chromatography-mass spectrometry (GC-MS) [3,4,6,24,25], liquid chromatography (LC)-MS, is currently taking a dominant role in their analysis [5,26]

Summary

Introduction

Sterols represent one of the major classes of lipids found in living systems [1]. In mammals, cholesterol represents the archetypal sterol. For decades there was little interest in these intermediates, in recent years the situation has changed with the realization that intermediates in bile acid biosynthesis are ligands to nuclear receptors, including the liver X receptors (LXRs, NR1H3, NR1H2) [10,11,12,13], farnesoid X receptor (FXR, NR1H4) [14], pregnane X receptor (PXR, known as xenobiotic sensing nuclear receptor, SXR, NR1I2) [15,16], RAR-related orphan receptor γt (RORγt, NR1F3) [17], and estrogen receptors (ERs, NR3A1, NR3A2) [18] They are related to G protein-coupled receptors (e.g., Epstein-Barr virus induced gene 2 (EBI2, GPR183) [19,20] and smoothened (SMO, FZD11) [21,22]), and are involved in the regulation of cholesterol biosynthesis by binding to INSIG1 (insulin induced gene 1) [23]. Cholesterol metabolites have traditionally been analyzed by gas-chromatography-mass spectrometry (GC-MS) [3,4,6,24,25], liquid chromatography (LC)-MS, is currently taking a dominant role in their analysis [5,26]

Methods

Results

Conclusion