24S-hydroxycholesterol in plasma: A marker of cholesterol turnover in neurodegenerative diseases

Brain cholesterol is mainly involved in the cell membrane structure, in signal transduction, neurotransmitter release, synaptogenesis and membrane trafficking. Impairment of brain cholesterol metabolism was described in neurodegenerative diseases, such as Multiple Sclerosis, Alzheimer and Huntington Diseases. Since the blood–brain barrier efficiently prevents cholesterol uptake from the circulation into the brain, de novo synthesis is responsible for almost all cholesterol present there. Cholesterol is converted into 24S-hydroxycholesterol (24OHC) by cholesterol 24-hydroxylase (CYP46A1) expressed in neural cells.     

The effect of oxysterols on the interaction of Alzheimer's amyloid beta with model membranes

The interaction of amyloid beta (Aβ) peptide with cell membranes has been shown to be influenced by Aβ conformation, membrane physicochemical properties and lipid composition. However, the effect of cholesterol and its oxidized derivatives, oxysterols, on Aβ-induced neurotoxicity to membranes is not fully understood. We employed here model membranes to investigate the localization of Aβ in membranes and the peptide-induced membrane dynamics in the presence of cholesterol and 7-ketocholesterol (7keto) or 25-hydroxycholesterol (25OH). Our results have indicated that oxysterols rendered membranes more sensitive to Aβ, in contrast to role of cholesterol in inhibiting Aβ/membrane interaction. We have demonstrated that two oxysterols had different impacts owing to distinct positions of the additional oxygen group in their structures. 7keto-containing cell-sized liposomes exhibited a high propensity toward association with Aβ, while 25OH systems were more capable of morphological changes in response to the peptide. Furthermore, we have shown that 42-amino acid Aβ (Aβ-42) pre-fibril species had higher association with membranes, and caused membrane fluctuation faster than 40-residue isoform (Aβ-40). These findings suggest the enhancing effect of oxysterols on interaction of Aβ with membranes and contribute to clarify the harmful impact of cholesterol on Aβ-induced neurotoxicity by means of its oxidation.     

Oxysterols induce transition of monocytic cells to phenotypically mature dendritic cell-like cells

Dendritic cells (DCs) activate adaptive immune responses in atherosclerotic plaques; however, the origin of DCs is in question. We attempted to determine whether cholesterol or its oxide forms, which are detected in abundance in atheromatous lesions, could induce differentiation or transition of monocytic cells to DCs. Treatment of THP-1 cells with 27-hydroxycholesterol (27OH-Chol) and 7α-hydroxycholesterol (7αOH-Chol) resulted in an increase in the numbers of adherent cells, and, in contrast to PMA, decreased uptake of FITC-conjugated dextran. In addition, treatment with 27OH-Chol and 7αOH-Chol induced expression of mDC-specific molecules, including CD40, CD80, CD83, and CD88. Of the two oxysterols, 27OH-Chol enhanced expression of MHC class I and II molecules as well as CCR7. However, treatment with an identical concentration of cholesterol and 7-ketocholesterol did not influence adherence, uptake of FITC-conjugated dextran, and expression of the aforementioned molecules. This is the first study to report on change of monocytic cells by oxysterols to phenotypically atypical cells with some characteristics of mDCs detected in atherosclerotic lesions. We propose that a certain type of oxysterol would contribute to immune responses in atherosclerotic lesions by enhancing expression of multiple CD molecules as well as MHC molecules by monocytic cells.     

Measurement of oxysterols and alpha-tocopherol in plasma and tissue samples as indices of oxidant stress status

Oxidant stress seems to play a role in several setting of human pathology, such as atherosclerosis, cancer, and aging. The study of oxidant stress in human disease should be based on the evaluation of either sensitive and specific markers of enhanced oxidant stress, such as oxysterols, or antioxidant defense, by measuring alpha-tocopherol. We have developed a rapid method to measure the oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol in plasma (50 healthy subjects) and tissue as an index of oxidant stress in vivo, and from the same sample alpha-tocopherol content. The mean plasma concentration of 7beta-hydroxycholesterol and 7-ketocholesterol was 4.6+/-1.1 and 13.4+/-7.6 ng/mL, respectively. Plasma alpha-tocopherol concentration was 5.8+/-1.0 micromol/mol cholesterol. Samples from atherosclerotic plaques contained 20 times more cholesterol, about 45 times higher oxysterols levels, and 600 times more alpha-tocopherol compared to normal arteries. No significant difference in cholesterol and oxysterol content was observed between cirrhotic and normal liver. However, cirrhotic liver contained significantly smaller concentration of alpha-tocopherol compared to normal liver. In conclusion, we have developed a rapid and reliable method for the assay of cholesterol oxidation products and alpha-tocopherol in plasma and tissue useful for estimation of oxidant stress/antioxidant balance.     

Potential of gas chromatography–atmospheric pressure chemical ionization–time-of-flight mass spectrometry for the determination of sterols in human plasma

The application of Gas Chromatography (GC)–Atmospheric Pressure Chemical Ionization (APCI)–Time-of-Flight Mass Spectrometry (TOF-MS) is presented for sterol analysis in human plasma. A commercial APCI interface was modified to ensure a well-defined humidity which is essential for controlled ionization. In the first step, optimization regarding flow rates of auxiliary gases was performed by using a mixture of model analytes. Secondly, the qualitative and quantitative analysis of sterols including oxysterols, cholesterol precursors, and plant sterols as trimethylsilyl-derivatives was successfully carried out. The characteristics of APCI together with the very good mass accuracy of TOF-MS data enable the reliable identification of relevant sterols in complex matrices. Linear calibration lines and plausible results for healthy volunteers and patients could be obtained whereas all mass signals were extracted with an extraction width of 20 ppm from the full mass data set. One advantage of high mass accuracy can be seen in the fact that from one recorded run any search for m/z can be performed.     

Short synthesis of 16beta-hydroxy-5alpha-cholestane-3,6-dione a novel cytotoxic marine oxysterol

The first and short synthesis of 16beta-hydroxy-5alpha-cholestane-3,6-dione 1 a metabolite from marine algae, has been achieved in six steps from readily available diosgenin 5. Selective deoxygenation of primary alcohol of triol 6 has been accomplished in one step using Et(3)SiH and catalytic amount of B(C(6)F(5))(3) to produce compound 9 in high yield. Oxidation of 11 with PCC, allowed the introduction of 3,6-ene-dione functionality, and further catalytic hydrogenation and deprotection furnished the 3,6-diketo steroid 1.     

Gas chromatography–mass spectrometry-based simultaneous quantitative analytical method for urinary oxysterols and bile acids in rats

A simultaneous quantitative assay method for urinary oxysterols and bile acids using GC–MS was developed to investigate the mechanism of liver toxicity induced by drugs or chemicals. Sample preparations were optimized by exploring various extraction solvents, derivatization reagents, and hydrolysis methods to achieve reliable and maximum sensitivity for these two different compound classes. As a result, satisfactory accuracy, precision, and sensitivity were obtained in the validation. The method was then applied to quantify urinary oxysterols and bile acids produced from liver toxicity induced by atorvastatin (250 mg/kg/day). From the results, increases in bile acid levels and decreases in the concentration ratio between cholic acid and chenodeoxycholic acid, which are the distinguishing phenomena observed in serum or bile for liver toxicity, were also observed in urine. Additionally, the mechanism of liver toxicity was investigated with the urinary concentration ratio of product to precursor in the metabolic pathway from cholesterol to bile acids. The results indicated that enzyme activities related to the production and degradation of bile acids, not oxysterols, were significantly changed from liver toxicity. Thus, it was concluded that urinary levels of oxysterols and bile acids could be useful tools for checking liver toxicity and investigating its mechanism.     

MAPK signaling in H9c2 cardiomyoblasts exposed to cholesterol secoaldehyde – Role of hydrogen peroxide

3β-Hydroxy-5,6-secocholestan-6-al (cholesterol secoaldehyde or ChSeco), an oxysterol known to be formed in ozone- and singlet oxygen-mediated oxidations of cholesterol, has been detected in the atherosclerotic plaque and in the brain of patients suffering from Alzheimer’s disease and Lewy body dementia. Previously, we have shown that, in H9c2 cardiomyoblasts, ChSeco induces oxidative stress followed by apoptosis involving both intrinsic and extrinsic signaling pathways. In the present study, we investigated the nature of reactive oxygen species (ROS) and its associated redox signaling in H9c2 cells upon treatment with ChSeco. Both catalase and deferoxamine, which lowered intracellular ROS, were found to alleviate the ChSeco-induced cytotoxicity. ChSeco-treated H9c2 cells showed a significant decrease in the intracellular catalase activity, suggesting the involvement of H₂O₂ in the associated cytotoxicity. Additionally, in ChSeco-exposed cells, there was a marked increase in lipid peroxidation and pre-treatment with SB 203580 (p38 MAPK inhibitor) and MEK1/2 inhibitor (ERK1/2 and JNK inhibitor) rendered protection against the cytotoxicity. An early increase in the expression of p-SAPK/JNK or delayed p38 MAPK did not alter ATF-2 but decreased c-Jun expression in these cells. Overall, these findings are consistent with MAPK signaling resulting from increased cellular H₂O₂ in ChSeco-induced cytotoxicity in cardiomyoblasts.     

Lipokines and oxysterols: Novel adipose-derived lipid hormones linking adipose dysfunction and insulin resistance

The expansion of adipose tissue (AT) is, by definition, a hallmark of obesity. However, not all increases in fat mass are associated with pathophysiological cues. Indeed, whereas a “healthy” fat mass accrual, mainly in the subcutaneous depots, preserves metabolic homeostasis, explaining the occurrence of the metabolically healthy obese phenotype, “unhealthy” AT expansion is importantly associated with insulin resistance/type 2 diabetes and the metabolic syndrome. The development of a dysfunctional adipose organ may find mechanistic explanation in a reduced ability to recruit new and functional (pre)adipocytes from undifferentiated precursor cells. Such a failure of the adipogenic process underlies the “AT expandability” paradigm. The inability of AT to expand further to store excess nutrients, rather than obesity per se, induces a diabetogenic milieu by promoting the overflow and the ectopic deposition of fatty acids in insulin-dependent organs (i.e., lipotoxicity), the secretion of various metabolically detrimental adipose-derived hormones (i.e., adipokines and lipokines), and the occurrence of local and systemic inflammation and oxidative stress. Hitherto, fatty acids (i.e., lipokines) and the oxidation by-products of cholesterol and polyunsaturated fatty acids, such as nonenzymatic oxysterols and reactive aldehyde species, respectively, emerge as key modulators of (pre)adipocyte signaling through Wnt/β-catenin and MAPK pathways and potential regulators of glucose homeostasis. These and other mechanistic insights linking adipose dysfunction, oxidative stress, and impairment of glucose homeostasis are discussed in this review article, which focuses on adipose peroxidation as a potential instigator of, and a putative therapeutic target for, obesity-associated metabolic dysfunctions.     

7-Ketocholesterol as marker of cholesterol oxidation in model and food systems: When and how

Cholesterol can undergo oxidation through enzymatic or chemical mechanisms, generating a wide range of oxidation products (COPs) with adverse biological effects. COPs are characterized by different functional groups and are produced in different ratios or amounts, depending on the treatment and storage conditions. To follow the cholesterol oxidation process, 7-ketocholesterol (7-KC) has been often used as an oxidation marker in both model and food systems, since it is easily formed and is one of the most representative ring COPs. However, 7-KC does not always rise with increasing time/temperature conditions, especially in complex systems and high-protein or extensively processed foods. The following review provides a critical picture of the utilization of 7-KC as a cholesterol oxidation marker in model and food systems, focusing on the possible causes and effects of the different behaviours and trends, as well as on the advantages and disadvantages of using 7-KC when the extent of cholesterol oxidation is to be assessed.