Sterols and oxysterols in brain and the immune system.

This project investigates sterol and oxysterol content of murine brain and macrophages. Oxysterols are oxidised forms of cholesterol implicated in a wide array of biological functions. The compounds were analysed with LC-MS LTQ-Orbitrap high resolution system, which provides a highly sensitive and accurate tool for analysis of metabolites. We profiled a sterol content of newborn murine brain and identified a broad spectrum of oxysterols. Some of these compounds are implicated in neurogenesis, a number of other oxysterols derived from desmosterol were identified in brain tissue for the first time. We analysed murine model of human disease arising from reduced ability to produce cholesterol (SLOS). The sterol profile showed an altered level of cholesterol precursors and generally lowered concentration of oxysterols. Next, we moved to analysis of brain lipidome from animals with disrupted mechanisms of cholesterol metabolism. Cholesterol hydroxylase Cyp46al provides the mechanism of cholesterol removal from the brain. The study of the knock out mice did not reveal the existence of any compensatory mechanisms. The results showed mainly the reduction in cholesterol synthesis. We profiled a sterol content of Cyp27al-/- mouse brain. Cyp27al participates in bile acid synthesis. The steroid profile revealed changed pattern of mono- and polihydroxylated sterols suggesting an upregulation of an alternative pathway of bile acid synthesis. We also analysed the consequence of deficiency of another enzyme involved in bile acids synthesis Cyp7bl. In brain of Cyp7bl-/- mouse we found elevated levels of known Cyp7bl substrates, and increased concentration of other, putative substrates for this enzyme. The last experimental chapter concentrates on analysis of murine macrophages treated with interferon beta and gamma. The treatment induced an increased production of 25-hydroxycholesterol. This links sterol metabolism with mechanisms of immune defence

A quantitative LC-MS/MS method for analysis of mitochondrial -specific oxysterol metabolism

Oxysterols are critical regulators of inflammation and cholesterol metabolism in cells. They are oxidation products of cholesterol and may be differentially metabolised in subcellular compartments and in biological fluids. New analytical methods are needed to improve our understanding of oxysterol trafficking and the molecular interplay between the cellular compartments required to maintain cholesterol/oxysterol homeostasis. Here we describe a method for isolation of oxysterols using solid phase extraction and quantification by liquid chromatography-mass spectrometry, applied to tissue, cells and mitochondria. We analysed five monohydroxysterols; 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7 ketocholesterol and three dihydroxysterols 7α-24(S)dihydroxycholesterol, 7α-25dihydroxycholesterol, 7α-27dihydroxycholesterol by LC-MS/MS following reverse phase chromatography. Our new method, using Triton and DMSO extraction, shows improved extraction efficiency and recovery of oxysterols from cellular matrix. We validated our method by reproducibly measuring oxysterols in mouse brain tissue and showed that mice fed a high fat diet had significantly lower levels of 24S/25diOHC, 27diOHC and 7ketoOHC. We measured oxysterols in mitochondria from peripheral blood mononuclear cells and highlight the importance of rapid cell isolation to minimise effects of handling and storage conditions on oxysterol composition in clinical samples. In addition, in vitro cell culture systems, of THP-1 monocytes and neuronal-like SH-SH5Y cells, showed mitochondrial-specific oxysterol metabolism and profiles were lineage specific. In summary, we describe a robust and reproducible method validated for improved recovery, quantitative linearity and detection, reproducibility and selectivity for cellular oxysterol analysis. This method enables subcellular oxysterol metabolism to be monitored and is versatile in its application to various biological and clinical samples

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

Mining for Oxysterols in Cyp7b1−/− Mouse Brain and Plasma: Relevance to Spastic Paraplegia Type 5

Deficiency in cytochrome P450 (CYP) 7B1, also known as oxysterol 7α-hydroxylase, in humans leads to hereditary spastic paraplegia type 5 (SPG5) and in some cases in infants to liver disease. SPG5 is medically characterized by loss of motor neurons in the corticospinal tract. In an effort to gain a better understanding of the fundamental biochemistry of this disorder, we have extended our previous profiling of the oxysterol content of brain and plasma of Cyp7b1 knockout (-/-) mice to include, amongst other sterols, 25-hydroxylated cholesterol metabolites. Although brain cholesterol levels do not differ between wild-type (wt) and knockout mice, we find, using a charge-tagging methodology in combination with liquid chromatography–mass spectrometry (LC–MS) and multistage fragmentation (MSn), that there is a build-up of the CYP7B1 substrate 25-hydroxycholesterol (25-HC) in Cyp7b1-/- mouse brain and plasma. As reported earlier, levels of (25R)26-hydroxycholesterol (26-HC), 3β-hydroxycholest-5-en-(25R)26-oic acid and 24S,25-epoxycholesterol (24S,25-EC) are similarly elevated in brain and plasma. Side-chain oxysterols including 25-HC, 26-HC and 24S,25-EC are known to bind to INSIG (insulin-induced gene) and inhibit the processing of SREBP-2 (sterol regulatory element-binding protein-2) to its active form as a master regulator of cholesterol biosynthesis. We suggest the concentration of cholesterol in brain of the Cyp7b1-/- mouse is maintained by balancing reduced metabolism, as a consequence of a loss in CYP7B1, with reduced biosynthesis. The Cyp7b1-/- mouse does not show a motor defect; whether the defect in humans is a consequence of less efficient homeostasis of cholesterol in brain has yet to be uncovered

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)

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

Sterols and oxysterols in plasma from Smith-Lemli-Opitz syndrome patients

Smith-Lemli-Opitz syndrome (SLOS) is a severe autosomal recessive disorder resulting from defects in the cholesterol synthesising enzyme 7-dehydrocholesterol reductase (Δ7-sterol reductase, DHCR7, EC 1.3.1.21) leading to a build-up of the cholesterol precursor 7-dehydrocholesterol (7-DHC) in tissues and blood plasma. Although the underling enzyme deficiency associated with SLOS is clear there are likely to be multiple mechanisms responsible for SLOS pathology. In an effort to learn more of the aetiology of SLOS we have analysed plasma from SLOS patients to search for metabolites derived from 7-DHC which may be responsible for some of the pathology. We have identified a novel hydroxy-8-dehydrocholesterol, which is either 24- or 25-hydroxy-8-dehydrocholesterol and also the known metabolites 26-hydroxy-8-dehydrocholesterol, 4-hydroxy-7-dehydrocholesterol, 3β,5α-dihydroxycholest-7-en-6-one and 7α,8α-epoxycholesterol. None of these metabolites are detected in control plasma at quantifiable levels (0.5 ng/mL)

The role of cholesterol metabolism and various steroid abnormalities in autism spectrum disorders : A hypothesis paper

© 2017 The Authors Autism Research published by Wiley Periodicals, Inc. on behalf of International Society for Autism Research.Peer reviewedPublisher PD