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Hundreds of NPC1 variants cause highly heterogeneous phenotypes. This study aims to explore the genotype–phenotype correlation of NPC1, especially for missense variants. In a well‐characterized cohort, phenotypes are graded into three clinical forms: mild, intermediate, and severe. Missense residue structural location was stratified into three categories: surface, partially, and fully buried. The association of phenotypes with the topography of the amino acid substitution in the protein structure was investigated in our cohort and validated in two reported cohorts. One hundred six unrelated NPC1 patients were enrolled. A significant correlation of genotype–phenotype was found in 81 classified individuals with two or one (the second was null variant) missense variant (p < 0.001): of 25 patients with at least one missense variant of surface (group A), 19 (76%) mild, six (24%) intermediate, and none severe; of 31 cases with at least one missense variant of partially buried without surface variants (group B), 11 (35%) mild, 16 (52%) intermediate, and four (13%) severe; of the remaining 25 patients with two or one buried missense variants (group C), eight (32%) mild, nine (36%) intermediate, and eight (32%) severe. Additionally, 7‐ketocholesterol, the biomarker, was lower in group A than in group B (p = 0.024) and group C (p = 0.029). A model was proposed that accurately predicted phenotypes of 72 of 90 (80%), 73 of85 (86%), and 64 of 69 (93%) patients in our cohort, Italian, and UK cohort, respectively. This study proposed a novel genotype–phenotype correlation in NPC1, linking the underlying molecular pathophysiology with clinical phenotype and aiding genetic counseling and evaluation in clinical practice. [ABSTRACT FROM AUTHOR]

Age‐related macular degeneration (AMD) is associated with formation of drusen, clusters of lipids, and oxidized lipid products under the retinal pigment epithelium (RPE). 7‐Ketocholesterol (7KC) is a form of oxidized cholesterol present in drusen and is hypothesized to play a role in AMD pathogenesis. The association of 7KC with cellular toxicity and inflammation, key elements of AMD pathology, has been demonstrated. However, the effects of 7KC on altering RPE bioenergetics, a potentially important pathologic process in AMD, are unclear. Herein, we describe the effects of non‐lethal doses of 7KC on the bioenergetics and phenotype of RPE cells in culture. Metabolic analysis demonstrated a significant dose‐dependent increase in total ATP production rates that was driven primarily by an increase in glycolysis. The increase in glycolysis was accompanied by an increase in glucose uptake and increased expression of hexokinase 1. Increased levels of Translocase of Outer Mitochondrial Membrane 20 and NADH:Ubiquinone Oxidoreductase Core Subunit S1, Succinate dehydrogenase, Ubiquinol‐Cytochrome C Reductase Core Protein 2, Cytochrome C Oxidase II, and ATP synthase subunit beta, proteins involved in oxidative phosphorylation (OXPHOS), were also seen. However, specific electron transport chain activity remained unchanged. 7KC‐treated cells also demonstrated a change in cellular morphology with decreased expression of epithelial markers. In summary, 7KC has significant effects on the bioenergetics and morphology of RPE cells reflective of findings seen in clinical AMD. [ABSTRACT FROM AUTHOR]

Summary: Clitoria ternatea (CT) petals have a high content of polyphenolic compounds, including anthocyanins, which protect against lipid oxidation. This research evaluated total phenolic content, total anthocyanin content and antioxidant activity of CT extracts obtained using distilled water, methanol, and/or water‐methanol combination (1:1) solvents after 6, 12 and 24 h soaking times. The predominant bioactive compounds of CT petals were kaempferol 3‐neohesperidoside (462.63 mg per 100 g), caffeoylmalic acid (137.59 mg per 100 g), and kaempferol 3‐(2G‐rhamnosylrutinoside) (129.28 mg per 100 g). The capacity of each solvent‐type extract obtained from 6 h soaking time to inhibit cholesterol oxidation was determined after 24 and 48 h in an emulsion model. At 6 h soaking time, the combination‐solvent yielded an extract with the highest anthocyanin content (63.9 μg mL−1) and inhibited 89.8% of 7‐ketocholesterol production in emulsion, compared to the control. This study demonstrated that CT petal extracts can enhance health benefits and lengthen shelf life of emulsion‐type products. Ability of Clitoria ternatea petal extracts to inhibit cholesterol oxidation (7‐KC) in an emulsion after 24 and 48 hours. [ABSTRACT FROM AUTHOR]

Cholesterol plays an important role in the interaction of Alzheimer’s amyloid beta (Aβ) with cell membranes, an important event in Aβ-induced cytotoxicity. However, it is not fully understood how cholesterol influences the association of Aβ with membrane lateral compartments. We have shown that by modulating membrane fluidity, cholesterol decreased peptide localization in solid-ordered domains and increased that in liquid-ordered domains. It changed the amount of Aβ associating with liquid-disordered (Ld) phase with different tendencies depending on the composition of heterogeneous membrane systems. 7-Ketocholesterol, an oxidized derivative of cholesterol, majorly enhanced the fluidity of and Aβ interaction with Ld phase. These findings are useful for clarifying the impact of cholesterol and its oxidation in Aβ-induced toxicity. [ABSTRACT FROM AUTHOR]

7-Ketocholesterol (7KC) is a cytotoxic oxysterol that plays a role in many age-related degenerative diseases. 7KC formation and accumulation often occurs in the lysosome, which hinders enzymatic transformations that reduce its toxicity and increase the sensitivity to lysosomal membrane permeabilization. We assayed the potential to mitigate 7KC cytotoxicity and enhance cell viability by overexpressing 7KC-active enzymes in human fibroblasts. One of the enzymes tested, a cholesterol oxidase engineered for lysosomal targeting, significantly increased cell viability in the short term upon treatment with up to 50 µM 7KC relative to controls. These results suggest targeting the lysosome for optimal treatment of oxysterol-mediated cytotoxicity, and support the use of introducing novel catalytic function into the lysosome for therapeutic and research applications. Biotechnol. Bioeng. 2012;109: 2409-2415. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The growth of the necrotic core region within advanced atherosclerotic plaque is thought to be driven by oxidised low density lipoprotein (oxLDL)-induced death of macrophage cells. OxLDL and atherosclerotic plaque are rich in oxysterols, especially 7-ketocholesterol (7KC). As 7KC triggers cell death at physiological concentrations when added directly to the cell culture media, 7KC and other oxysterols have been suggested to be the main cytotoxic agent of oxLDL. We investigated this hypothesis by examining the toxicity of 7KC to monocyte-like U937 cells when incorporated into high-uptake non-toxic acetylated LDL (acLDL). Incorporation of 7KC into acLDL greatly reduced the oxysterol toxicity when compared with an equivalent amount of 7KC added directly to U937 cells. Enrichment of oxLDL with 7KC did not significantly enhance lipoprotein toxicity. OxLDL was highly cytotoxic yet generated only low levels of intracellular 7KC. In comparison, 7KC-acLDL generated high intracellular 7KC concentrations with little loss in cell viability. The data show that when incorporated into lipoprotein, 7KC cytotoxicity is greatly reduced, even though intracellular levels exceed those measured when cells are incubated with oxLDL, which suggests 7KC is not the significant toxic agent within oxLDL. [ABSTRACT FROM AUTHOR]

The present study was carried out to elucidate changes in the gene expression and activity of cholesterol biosynthetic enzymes and transporters in the rat hippocampus after kainate excitotoxicity. Significantly increased cholesterol level was detected in the degenerating hippocampus, reaching double normal levels at 1 week after kainate injury. RT-PCR analyses of hippocampal homogenates showed significantly decreased mRNA expression of the transcription factor controlling cholesterol biosynthesis SREBP-2, and the rate-controlling enzyme HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase at all time points after kainate injection; and decreased lanosterol synthase and CYP51 at 1 and 2 weeks post-kainate injection respectively. GC-MS analyses showed a significant increase in cholesterol biosynthetic precursors lanosterol, desmosterol and 7-dehydrocholesterol at 1 day after kainate injection presumably reflecting biosysnthesis in injured neurons, and significant decreases in precursors at 1 and 2 weeks post-kainate injection, at time of gliosis in the degenerating hippocampus. Levels of cholesterol autooxidation including 7 ketocholesterol and cholesterol epoxides were elevated in the kainate lesioned hippocampus. Furthermore, loss of expression of the cholesterol transporter, ABCA1 was detected in neurons, but increased expression in astrocytes was detected after kainate lesions. The results suggest that increased cholesterol biosynthesis and loss of ABCA1 expression in injured neurons might result in increase in cholesterol in the degenerating hippocampus. The increased cholesterol might predispose to increased formation of cholesterol oxidation products which have been shown to be toxic to neurons. [ABSTRACT FROM AUTHOR]

Examines the effect of processing time and temperature on the occurrence of cholesterol oxidation products (COP) in butter. Heating of butter for different periods of time at different temperatures relevant to the conditions of daily cooking; Finding that COP were barely detectable in butter; Impact of process temperature and time on the amount of COP.

Oxysterols, and particularly 7-ketocholesterol, appear to be strongly involved in the physiopathology of atherosclerosis. These molecules are suspected to be cytotoxic to the cells of the vascular wall and monocytes/macrophages, particularly by inducing apoptosis. Previous studies have demonstrated that 7-ketocholesterol-induced apoptosis is triggered by a sustained increase of cytosolic-free Ca2+, which elicits the mitochondrial pathway of apoptosis by activation of the calcium-dependent phosphatase calcineurin, leading to dephosphorylation of the‘BH3 only’ protein BAD. However, thorough study of the results suggests that other pathways are implicated in 7-ketocholesterol-induced cytotoxicity. In this study, we demonstrate the involvement of two other calcium-dependent pathways during 7-ketocholesterol-induced apoptosis. The activation of the MEK→ERK pathway by the calcium-dependent tyrosine kinase PYK 2, a survival pathway which delays apoptosis as shown by the use of the MEK inhibitor U0126, and a pathway involving another pro-apoptotic BH3 only protein, Bim. Indeed, 7-ketocholesterol treatment of human monocytic THP-1 cells induces the release of Bim-LC8 from the microtubule-associated dynein motor complex, and its association with Bcl-2. Therefore, it appears that 7-ketocholesterol-induced apoptosis is a complex phenomenon resulting from calcium-dependent activation of several pro-apoptotic pathways and also one survival pathway. [ABSTRACT FROM AUTHOR]

The potential of various cooking oils to prevent k cholesterol degradation and/or oxidation, as measured by the production of 7-ketocholesterol during heating at different temperatures, was studied using a cholesterol model system. In the control group (without cooking oil), cholesterol was relatively stable, and 73% of its initial concentration was present after 30 min of heating at 1257deg;C. Less than 30 and 10% of cholesterol remained at 150 and 175°C after 30 min, respectively, and 10% at 200°C after 10 min. In the treatment group, cholesterol mixed with corn, canola, soybean, or olive oil had significantly improved thermal stability. More than 60 and 40% of cholesterol remained at 150 and 175°C after 30 min, respectively. In the control group, 7-ketocholesterol was produced when samples were heated above 150°C, and levels increased consistently during 30 min of heating. At 175 or 200°C, the level of 7-ketocholesterol did not increase further after reaching the highest level after 10 min of heating. 7-Ketocholesterol is not stable above 175°C, and its degradation rate could be much faster than its production at 200°C. 7-Ketocholesterol was not found in samples of cholesterol mixed with corn oil or laboratory-prepared soybean and rice bran oils until the heating temperature was raised to 175°C for 20 min. The levels of 7-ketocholesterol in those treatment groups were greater than that in the control group at 175°C for 30 min. These oils may increase the thermal stability of 7-ketocholesterol and retard its degradation rate. [ABSTRACT FROM AUTHOR]

The inhibition of cholesterol autoxidation by nonsaponifiable fraction from rice bran oil (700, 1400, and 2100 ppm) was studied in an aqueous model system for 16 h at pH 5.5 and 80°C. Antioxidant effectiveness was investigated by following the loss of cholesterol and the formation of 7-keto-cholesterol. The changes in levels of vitamin E vitamers and γ-oryzanol in the system were determined during cholesterol autoxidation. The 2100 ppm treatment produced a 92% reduction of 7-ketocholesterol, 1400 ppm an 82% reductions and 700 ppm a 64% reduction after 16 h, whereas without the nonsaponifiable fraction, the samples showed almost complete degradation of cholesterol. Vitamin E vitamers decreased ( P<0.05) in all treatments, but γ-oryzanol was not significantly ( P<0.05) reduced in the 2100 ppm treatment. [ABSTRACT FROM AUTHOR]

Two methods for routine analysis of free 7-ketocholesterol (7-k) in foods by high-performance liquid chromatography (HPLC) were compared. The analyzed samples were egg noodles, biscuits prepared with eggs, sweet snacks, grated Parmesan cheeses, and some ingredients utilized in the food industry (whole-milk and whole-egg powder). The enrichment of cholesterol oxides was carried out by solid-phase separation of the total lipids with florisil and silica cartridges for methods A and B, respectively. The 7-k analyses were run in normal-phase HPLC for method A, and a reverse-phase process was used in method B. Identification of the 7-k peak was confirmed by gas chromatography/mass spectrometry analysis and peak purity check via spectral analysis with a diode array detector. The quantitation of 7-k was carried out with an internal standard for method A and with a calibration curve for method B. The limit of quantitation (LQ) was 3 × 10 g/injection; the limit of detection was ten times lower than the LQ for both methods. The two methods showed good recovery (99%) and good repeatability (coefficient of variation of 3.9 and 3.7% for methods A and B, respectively). These methods allow a fast, sensitive, and reliable determination of one cholesterol oxidation product, which is present at a high concentration level in the first stages of the oxidation process. [ABSTRACT FROM AUTHOR]

In order to evaluate the effects of the degree of unsaturation of triacylglycerols on cholesterol oxidation, mixtures of purified sardine oil triacylglycerols (iodine value, IV=182.6) and cholesterol; of partially hydrogenated sardine oil triacylglycerols (IV=174.5) and cholesterol; and of fully hydrogenated sardine oil triacylglycerols (IV=92.0) and cholesterol were incubated at 25°C in the dark. The oxidative stability of the samples decreased with increasing degree of unsaturation of the triacylglycerols in the sample mixtures; the induction period for peroxide values (PV) of the sardine oil triacylglycerols and cholesterol was shorter than that of the partially hydrogenated sardine oil triacylglycerols and cholesterol. Certain polyunsaturated fatty acids (PUFAs) in the constituent fatty acids of sardine oil triacylglycerols started to decrease after a shorter induction period compared with that of the partially hydrogenated triacylglycerols. The prominent cholesterol oxides accumulated in the samples were 7β-hydroxycholesterol, 7-ketocholesterol, β-epoxide and cholestane triol. The tendency for accumulation of cholesterol oxides in the time course coincided with the changes in PV as well as the decrease in PUFAs. Cholesterol was oxidized in conjunction with autoxidation of coexisting fish oil triacylglycerols. Although lowering the degree of unsaturation of fish oil triacylglycerols was effective in prolonging the induction period of cholesterol oxidation, the rate of cholesterol oxidation in the cholesterol oxides' formation phase after the induction period was not affected by the difference in the proportion of highly unsaturated fatty acids in the natural and partially hydrogenated triacylglycerols of fish oils. [ABSTRACT FROM AUTHOR]

7-Ketocholesterol in aqueous sodium stearate dispersions or incorporated into liposomes was exposed to gamma radiation at 0-4°C to a dose of 50 kGy. Substrate degradation was noted in all systems, but occurred to a much greater extent in dispersions (≈90%) than in liposomes (0-25%). The difference in degree of degradation is attributed to dissimilarities in the micro-environment of 7-ketocholesterol in the two systems. The principal decomposition product was identified as 7-ketocholestanol, a compound that can arise from hydrogenation of the double bond of the starting material. Also detected, but in lower concentration, were the epimeric 7-hydroxycholesterols resulting from the reduction of the original carbonyl function. Both 7-ketocholestanol and the epimeric 7-hydroxycholesterols are believed to be secondary species generated by the interaction of the starting material with the primary radiolysis products of water. In addition, many minor degradation products were noted but not identified. [ABSTRACT FROM AUTHOR]