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

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

Charge-tagging liquid chromatography–mass spectrometry methodology targeting oxysterol diastereoisomers

The introduction of a hydroxy group to the cholesterol skeleton introduces not only the possibility for positional isomers but also diastereoisomers, where two or more isomers have different configurations at one or more of the stereocentres but are not mirror images. The differentiation of diastereoisomers is important as differing isomers can have differing biochemical properties and are formed via different biochemical pathways. Separation of diasterioisomers is not always easy by chromatographic methods. Here we demonstrate, by application of charge-tagging and derivatisation with the Girard P reagent, the separation and detection of biologically relevant diastereoisomers using liquid chromatography – mass spectrometry with multistage fragmentation

24(S),25-Epoxycholesterol and cholesterol 24S-hydroxylase (CYP46A1) overexpression promote midbrain dopaminergic neurogenesis in vivo

The liver X receptors Lxrα/NR1H3 and Lxrβ/NR1H2 are ligand-dependent nuclear receptors critical for midbrain dopaminergic (mDA) neuron development. We previously found that 24(S),25-epoxycholesterol (24,25-EC), the most potent and abundant Lxr ligand in the developing mouse midbrain, promotes mDA neurogenesis in vitro. In this study, we demonstrate that 24,25-EC promotes mDA neurogenesis in an Lxr-dependent manner, in the developing mouse midbrain in vivo and also prevents toxicity induced by the Lxr inhibitor geranylgeranyl pyrophosphate. Furthermore, using MS we show that overexpression of human cholesterol 24S-hydroxylase (CYP46A1) increases the levels of both 24(S)-hydroxycholesterol (24-HC) and 24,25-EC in the developing midbrain, resulting in a specific increase in mDA neurogenesis in vitro and in vivo, but has no effect on occulomotor or red nucleus neurogenesis. 24-HC, unlike 24,25-EC, did not affect in vitro neurogenesis, indicating that the neurogenic effect of 24,25-EC on mDA neurons is specific. Combined, our results indicate that increased levels of 24,25-EC in vivo, by intracerebroventricular delivery in wild-type mice or by overexpression of its biosynthetic enzyme CYP46A1, specifically promote mDA neurogenesis. We propose that increasing the levels of 24,25-EC in vivo may be a useful strategy to combat the loss of mDA neurons in Parkinson’s disease

International lease accounting reform and economic consequences: the views of UK users and preparers

In response to perceived difficulties with extant lease-accounting standards in operation worldwide, the G4+1 issued a discussion paper which proposes that all leases should be recognized on the balance sheet [ASB (1999). Leases: Implementation of a new approach, discussion paper. London: Accounting Standards Board]. Leasing is now on the active agenda of the IASB. A major difficulty faced by standard setters lies in overcoming the preparer/user lobbying imbalance and obtaining ex ante evidence on the likely impact of regulatory reform. This paper contributes to the ongoing international debate by conducting a questionnaire survey of U.K. users and preparers to assess their views on proposals for lease-accounting reform and on the potential economic consequences of their adoption. The results, based on 132 responses, indicate that both groups accept that there are deficiencies in the current rules, but they do not agree on the way forward and believe that the proposals would lead to significant economic consequences for key parties. The impact on respondents' views of familiarity with the proposals, level of lease usage, and company size, is also examined

Bile acid biosynthesis in Smith-Lemli-Opitz syndrome bypassing cholesterol: Potential importance of pathway intermediates

Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ5-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography – mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7β-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3β-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7β-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7β-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway

Identification of unusual oxysterols and bile acids with 7-oxo or 3β,5α,6β-trihydroxy functions in human plasma by charge-tagging mass spectrometry with multistage fragmentation

7-Oxocholesterol (7-OC), 5,6-epoxycholesterol (5,6-EC) and its hydrolysis product cholestane-3β,5α,6β-triol (3β,5α,6β-triol) are normally minor oxysterols in human samples, however, in disease their levels may be greatly elevated. This is the case in plasma from patients suffering from some lysosomal storage disorders e.g. Niemann Pick disease type C, or the inborn errors of sterol metabolism e.g. Smith-Lemli-Opitz syndrome and cerebrotendinous xanthomatosis. A complication in the analysis of 7-OC and 5,6-EC is that they can also be formed ex vivo from cholesterol during sample handling in air causing confusion with molecules formed in vivo. When formed endogenously 7-OC, 5,6-EC and 3β,5α,6β-triol can be converted to bile acids. Here, we describe methodology based on chemical derivatisation and liquid chromatography – mass spectrometry with multistage fragmentation (MSn) to identify the necessary intermediates in the conversion of 7-OC to 3β-hydroxy-7-oxochol-5-enoic acid and 5,6-EC and 3β,5α,6β-triol to 3β,5α,6β-trihydroxycholanoic acid. Identification of intermediate metabolites is facilitated by their unusual MSn fragmentation patterns. Semi-quantitative measurements are possible, but absolute values await the synthesis of isotope-labelled standards

Metabolism of Non-Enzymatically Derived Oxysterols: Clues from sterol metabolic disorders

Cholestane-3β,5α,6β-triol (3β,5α,6β-triol) is formed from cholestan-5,6-epoxide (5,6-EC) in a reaction catalysed by cholesterol epoxide hydrolase, following formation of 5,6-EC through free radical oxidation of cholesterol. 7-Oxocholesterol (7-OC) and 7β-hydroxycholesterol (7β-HC) can also be formed by free radical oxidation of cholesterol. Here we investigate how 3β,5α,6β-triol, 7-OC and 7β-HC are metabolised to bile acids. We show, by monitoring oxysterol metabolites in plasma samples rich in 3β,5α,6β-triol, 7-OC and 7β-HC, that these three oxysterols fall into novel branches of the acidic pathway of bile acid biosynthesis becoming (25R)26-hydroxylated then carboxylated, 24-hydroxylated and side-chain shortened to give the final products 3β,5α,6β-trihydroxycholanoic, 3β-hydroxy-7-oxochol-5-enoic and 3β,7β-dihydroxychol-5-enoic acids, respectively. The intermediates in these pathways may be causative of some phenotypical features of, and/or have diagnostic value for, the lysosomal storage diseases, Niemann Pick types C and B and lysosomal acid lipase deficiency. Free radical derived oxysterols are metabolised in human to unusual bile acids via novel branches of the acidic pathway, intermediates in these pathways are observed in plasma

Current trends in oxysterol research

In this short review we provide a synopsis of recent developments in oxysterol research highlighting topics of current interest to the community. These include the involvement of oxysterols in neuronal development and survival, their participation in the immune system, particularly with respect to bacterial and viral infection and to Th17-cell development, and the role of oxysterols in breast cancer. We also discuss the value of oxysterol analysis in the diagnosis of disease

New methods for analysis of oxysterols and related compounds by LC–MS

Oxysterols are oxygenated forms of cholesterol or its precursors. They are formed enzymatically and via reactive oxygen species. Oxysterols are intermediates in bile acid and steroid hormone biosynthetic pathways and are also bioactive molecules in their own right, being ligands to nuclear receptors and also regulators of the processing of steroid regulatory element-binding proteins (SREBPs) to their active forms as transcription factors regulating cholesterol and fatty acid biosynthesis. Oxysterols are implicated in the pathogenesis of multiple disease states ranging from atherosclerosis and cancer to multiple sclerosis and other neurodegenerative diseases including Alzheimer’s and Parkinson’s disease. Analysis of oxysterols is challenging on account of their low abundance in biological systems in comparison to cholesterol, and due to the propensity of cholesterol to undergo oxidation in air to generate oxysterols with the same structures as those present endogenously. In this article we review the mass spectrometry-based methods for oxysterol analysis paying particular attention to analysis by liquid chromatography – mass spectrometry (LC-MS)

The Cerebrospinal Fluid Profile of Cholesterol Metabolites in Parkinson’s Disease and Their Association With Disease State and Clinical Features

Disordered cholesterol metabolism is linked to neurodegeneration. In this study we investigated the profile of cholesterol metabolites found in the cerebrospinal fluid (CSF) of Parkinson’s disease (PD) patients. When adjustments were made for confounding variables of age and sex, 7α,(25R)26-dihydroxycholesterol and a second oxysterol 7α,x,y-trihydroxycholest-4-en-3-one (7α,x,y-triHCO), whose exact structure is unknown, were found to be significantly elevated in PD CSF. The likely location of the additional hydroxy groups on the second oxysterol are on the sterol side-chain. We found that CSF 7α-hydroxycholesterol levels correlated positively with depression in PD patients, while two presumptively identified cholestenoic acids correlated negatively with depression