The production of oxysterols in bile by activated human leukocytes

Oxysterols are naturally occurring intermediates in the conversion of cholesterol to bile acids, the major route for elimination of cholesterol. Additionally, they are important signaling agents, particularly in control of cholesterol synthesis; however, some species also are cytotoxic and carcinogenic. Oxysterols in plasma, contained in oxidized low-density lipoprotein, are strongly correlated with atherosclerosis. Oxysterols are found in infected human bile and the oxysterol content in gallstones correlates with bacterial DNA in the stones. Here we demonstrate that human leukocytes, activated by the presence of bacterial lipopolysaccharide, are able to oxidize cholesterol to a variety of oxysterols, including species known to be carcinogenic.     

Nano-liquid chromatography-tandem mass spectrometry analysis of oxysterols in brain: monitoring of cholesterol autoxidation

Oxysterols are present in mammalian brain at ng/g–μg/g levels while cholesterol is present at the mg/g level. This makes oxysterol analysis of brain challenging. In an effort to meet this challenge we have developed, and validated, an isolation method based on solid phase extraction and an analytical protocol involving oxidation/derivatisation (i.e., charge-tagging) followed by nano-flow liquid chromatography (nano-LC) combined with tandem mass spectrometry utilising multi-stage fragmentation (MSⁿ). The oxidation/derivatisation method employed improves detection limits by two orders of magnitude, while nano-LC–MSⁿ provides separation of isomers and allows oxysterol quantification. Using this method 13 different oxysterols have been identified in rat brain including 24S-hydroxycholesterol, 24S,25-epoxycholesterol and 7α,26-dihydroxycholest-4-en-3-one. The level of 24S-hydroxycholesterol in rat brain was determined to be 20.3 ± 3.4 μg/g and quantitative estimates were made for the other oxysterols identified. The presence of a large excess of cholesterol over oxysterol in brain raises the problem of autoxidation during sterol isolation and sample preparation. Thus, in parallel to identification studies, the degree of cholesterol autoxidation occurring during sterol isolation and analysis has been evaluated with the aid of [²H₇]-labelled cholesterol and cholesterol autoxidation products identified.     

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.     

Oxysterols: From cholesterol metabolites to key mediators

Oxysterols are cholesterol metabolites that can be produced through enzymatic or radical processes. They constitute a large family of lipids (i.e. the oxysterome) involved in a plethora of physiological processes. They can act through GPCR (e.g. EBI2, SMO, CXCR2), nuclear receptors (LXR, ROR, ERα) and through transporters or regulatory proteins. Their physiological effects encompass cholesterol, lipid and glucose homeostasis. Additionally, they were shown to be involved in other processes such as immune regulatory functions and brain homeostasis. First studied as precursors of bile acids, they quickly emerged as interesting lipid mediators. Their levels are greatly altered in several pathologies and some oxysterols (e.g. 4β-hydroxycholesterol or 7α-hydroxycholestenone) are used as biomarkers of specific pathologies. In this review, we discuss the complex metabolism and molecular targets (including binding properties) of these bioactive lipids in human and mice. We also discuss the genetic mouse models currently available to interrogate their effects in pathophysiological settings. We also summarize the levels of oxysterols reported in two key organs in oxysterol metabolism (liver and brain), plasma and cerebrospinal fluid. Finally, we consider future opportunities and directions in the oxysterol field in order to gain a better insight and understanding of the complex oxysterol system.     

Membrane and protein interactions of oxysterols

PURPOSE OF REVIEW: Oxysterols, oxidation products of cholesterol, mediate numerous and diverse biological processes. The objective of this review is to explain some of the biochemical and cell biological properties of oxysterols based on their membrane biophysical properties and their interaction with integral and peripheral membrane proteins. RECENT FINDINGS: According to their biophysical properties, which can be distinct from those of cholesterol, oxysterols can promote or inhibit the formation of membrane microdomains or lipid rafts. Oxysterols that inhibit raft formation are cytotoxic. The stereo-specific binding of cholesterol to sterol-sensing domains in cholesterol homeostatic pathways is not duplicated by oxysterols, and some oxysterols are poor substrates for the pathways that detoxify cells of excess cholesterol. The cytotoxic roles of oxysterols are, at least partly, due to a direct physical effect on membranes involved in cholesterol-induced cell apoptosis and raft mediated cell signaling. Oxysterols regulate cellular functions by binding to oxysterol binding protein and oxysterol binding protein-related proteins. Oxysterol binding protein is a sterol-dependent scaffolding protein that regulates the extracellular signal-regulated kinase signaling pathway. According to a recently solved structure for a yeast oxysterol binding protein-related protein, Osh4, some members of this large family of proteins are likely sterol transporters. SUMMARY: Given the association of some oxysterols with atherosclerosis, it is important to identify the mechanisms by which their association with cell membranes and intracellular proteins controls membrane structure and properties and intracellular signaling and metabolism. Studies on oxysterol binding protein and oxysterol binding protein-related proteins should lead to new understandings about sterol-regulated signal transduction and membrane trafficking pathways in cells.     

Increased serum oxysterol concentrations in patients with chronic hepatitis C virus infection

Oxidative stress and dysregulated cholesterol metabolism are characteristic features of chronic hepatitis C virus infection (CHC). Therefore, we analyzed serum oxysterol profiles in CHC patients and examined the significance of oxysterols in CHC. The concentrations of 7α-hydroxycholesterol, 4β-hydroxycholesterol and 25-hydroxycholesterol as determined by LC–ESI–MS/MS were significantly elevated by +236%, +29% and +44%, respectively, in CHC patients compared with controls. Moreover, the elevated levels were significantly decreased by anti-viral therapy using PEGylated-interferon and ribavirin for 3 months. In contrast, 24S-hydroxycholesterol, 27-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one concentrations were not affected by CHC or anti-viral treatment. These results suggest that some oxysterols that are elevated in CHC are produced by cholesterol autoxidation due to oxidative stress or inflammation in the liver. Oxysterols may represent novel targets for the inhibition of disease progression and the prevention of hepatocarcinogenesis in CHC patients.     

Evaluation of novel derivatisation reagents for the analysis of oxysterols

Oxysterols are oxidised forms of cholesterol that are intermediates in the synthesis of bile acids and steroid hormones. They are also ligands to nuclear and G protein-coupled receptors. Analysis of oxysterols in biological systems is challenging due to their low abundance coupled with their lack of a strong chromophore and poor ionisation characteristics in mass spectrometry (MS). We have previously used enzyme-assisted derivatisation for sterol analysis (EADSA) to identify and quantitate oxysterols in biological samples. This technique relies on tagging sterols with the Girard P reagent to introduce a charged quaternary ammonium group. Here, we have compared several modified Girard-like reagents and show that the permanent charge is vital for efficient MSⁿ fragmentation. However, we find that the reagent can be extended to include sites for potential stable isotope labels without a loss of performance.     

Oxysterol binding proteins: in more than one place at one time?

Oxysterols are potent signalling lipids that directly bind liver X receptors (LXRs) and a subset of oxysterol binding protein (OSBP) related proteins (ORPs). It is relatively well established that the oxysterol-regulated function of LXRs is to control the expression of genes involved in reverse cholesterol transport, catabolism of cholesterol, and lipogenesis. In contrast, the mechanisms by which oxysterols and ORPs affect cellular lipid metabolism have remained poorly understood. In this review, we summarize the information available on function of the ORPs and compare the two families of proteins binding oxysterol to demonstrate the different responses that similar lipids can elicit within cells. The other focus is on the membrane targeting determinants and the protein interaction partners of ORPs, which provide interesting clues to the mode(s) of ORP action. Specifically, we suggest a model in which a general property of ORPs is to function at membrane contact sites, specialized zones of communication between two different organelles.     

Cross-Talk between Oxysterols and Glucocorticoids: Differential Regulation of Secreted Phopholipase A2 and Impact on Oligodendrocyte Death

Background

Oxysterols are oxidized forms of cholesterol. They have been shown to be implicated in cholesterol turnover, inflammation and in neurodegenerative diseases such as Alzheimer''s disease and multiple sclerosis. Glial cells are targets of oxysterols: they inhibit astrocyte proliferation after brain injury, and we have previously shown that 25-hydroxycholesterol (25OH) provokes oligodendrocyte apoptosis and stimulates the expression of sPLA2 type IIA (sPLA2-IIA), which has a protective effect.

Methodology/Principal Findings

As glucocorticoids are well-known for their anti-inflammatory effects, our aim was to understand their direct effects on oxysterol-induced responses in oligodendrocytes (sPLA2-IIA stimulation and apoptosis). We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) abolishes the stimulation of sPLA2-IIA by 25-hydroxycholesterol (25-OH). This inhibition is mediated by the glucocorticoid receptor (GR), which decreases the expression of the oxysterol receptor Pregnane X Receptor (PXR) and interferes with oxysterol signaling by recruiting a common limiting coactivator PGC1α. Consistent with the finding that sPLA2-IIA can partially protect oligodendrocytes against oxysterol-triggered apoptosis, we demonstrate here that the inhibition of sPLA2-IIA by Dex accelerates the apoptotic phenomenon, leading to a shift towards necrosis. We have shown by atomic force microscopy and electron microscopy that 25-OH and Dex alters oligodendrocyte shape and disorganizes the cytoplasm.

Conclusions/Significance

Our results provide a new understanding of the cross-talk between oxysterol and glucocorticoid signaling pathways and their respective roles in apoptosis and oligodendrocyte functions.     

Oxysterols formation as a reflection of biochemical pathways: summary of in vitro and in vivo studies

Oxysterols are cholesterol (CH)-oxidized products generated in organs via either enzymatic or non-enzymatic pathways. Their presence or absence in cells, tissues and organs may provide information related to, for example, CH level and environmental status, inflammatory conditions near the CH molecules, activity of specific enzymes at and around the CH site, types and concentrations of biochemicals interacting with the CH, and the existence of specific signals. Here we present a mini-review of our lab findings on oxysterols formation in vitro and in vivo, including: the effects of different reactive species and availability of endogenous compounds on the type of oxysterol generated, the effects of enhanced activity of paraoxonase 1 or hemeoxygenase on oxysterol level, the correlation between human diseases such as diabetes and oxysterol accumulation, and the correlation between oxidative stress in neurons pre-Parkinsonian conditions in an animal model and intracellular oxidative stress.     

Simultaneous Determination of Oxysterols,Cholesterol and 25-Hydroxy-Vitamin D3 in Human Plasma by LC-UV-MS

Background

Oxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers.

Methods

A method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated.

Results

Our method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 μL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 μg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S)-, 25-, 27-, 7α-hydroxycholesterol (HC) and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis.

Conclusion

The method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential biomarkers of disease.     

Oxysterols in human circulation: which role do they have?

Oxysterols are oxygenated derivatives of cholesterol that are intermediates in cholesterol excretion pathways. They may also be regarded as transport forms of cholesterol and introduction of an additional hydroxyl group facilitates flux of cholesterol across cell membranes and the blood-brain barrier. According to current concepts, oxysterols are also mediating a number of cholesterol-induced metabolic effects. The recent discovery of nuclear receptors with an affinity for oxysterols has given support to this concept. Nuclear receptors such as liver X receptor alpha do have a role in cholesterol homeostasis, but there is still only indirect evidence that oxysterols are the physiological ligands. In this overview we report some recent advancements in our knowledge about the origin and metabolic fate of the quantitatively most important oxysterols occurring in the circulation. In addition, we discuss the possibility that some of these oxysterols may activate liver X receptors and regulate cholesterol homeostasis.     

Molecular Characterization of Oxysterol Binding to the Epstein-Barr Virus-induced Gene 2 (GPR183)

Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.     

25‐Hydroxycholesterol Acts in the Golgi Compartment to Induce Degradation of Tyrosinase

Oxysterols play a significant role in cholesterol homeostasis. 25‐Hydroxycholesterol (25HC) in particular has been demonstrated to regulate cholesterol homeostasis via oxysterol‐binding protein and oxysterol‐related proteins, the sterol regulatory element binding protein, and the rate‐limiting enzyme of cholesterol biosynthesis, hydroxymethylglutaryl coenzyme A reductase. We have examined the effect of 25HC on pigmentation of cultured murine melanocytes and demonstrated a decrease in pigmentation with an IC₅₀ of 0.34 μM and a significant diminution in levels of melanogenic protein tyrosinase. Pulse‐chase studies of 25HC‐treated cells demonstrated enhanced degradation of tyrosinase, the rate‐limiting enzyme of melanin synthesis, following endoplasmic reticulum (ER) and Golgi maturation. Protein levels of GS28, a member of an ER/cis‐Golgi SNARE protein complex, were also diminished in 25HC‐treated melanocytes, however levels of the ER chaperone calnexin and the cis‐Golgi matrix protein GM130 were unaffected. Effects of 25HC on tyrosinase were completely reversed by 4α‐allylcholestan‐3α‐ol, a sterol identified by its ability to reverse effects of 25HC on cholesterol homeostasis. Finally, the addition of 25HC to lipid deficient serum inhibited correct processing of tyrosinase. We conclude that 25HC acts in the Golgi compartment to regulate pigmentation by a mechanism shared with cholesterol homeostasis.