Cholesterol Oxidation Products Research Articles

7-Ketocholesterol enhances leukocyte adhesion to endothelial cells via p38MAPK pathway.

7-Ketocholesterol is a major dietary cholesterol oxidation product found in high concentrations in atherosclerotic plaques, which contribute to the development of atherosclerosis. This study aimed to investigate the effects of 7-ketocholesterol on endothelial inflammation, as well as the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with 7-ketocholesterol significantly enhanced the total interactions between human monocytic cells (THP-1 cell line) and TNFα-activated HUVECs under physiological flow conditions, compared to pretreatment with cholesterol (TNFα+50 μM cholesterol: 13.1 ± 0.54 cells/CPF, TNFα+50 μM 7-ketocholesterol: 18.9 ± 0.35 cells/CPF, p < 0.01). 7-Ketocholesterol enhanced the expression of E-selectin, ICAM-1, and VCAM-1 proteins. It also activated p38 mitogen-activated protein kinase (MAPK), and treatment with a p38 MAPK inhibitor inhibited both E-selectin expression via ATF-2 activation and 7-ketocholesterol-induced THP-1 adhesion to HUVECs. These findings suggest that 7-ketocholesterol enhances leukocyte–endothelial interactions by upregulating the expression of adhesion molecules, presumably via the p38 MAPK-dependent pathway.

Impairment of trophoblast survival and differentiation by LXR ligands is prevented by cholesterol but not ABCA1 silencing

IntroductionThe Liver X Receptors (LXRs) drive the transcriptional response to excess intracellular cholesterol. Oxysterols, the products of cholesterol oxidation, are activating ligands for LXR that can accumulate under conditions of oxidative stress and disrupt cholesterol homeostasis. While activation of LXR inhibits trophoblast differentiation, the impact of LXR on trophoblast physiology or cholesterol homeostasis is incompletely understood. We sought to determine if the effects of LXR activation can be ameliorated through modification of cholesterol bioavailability or inhibition of LXR-driven cholesterol efflux in trophoblasts. MethodsWe measured the effect of oxysterol exposure on BeWo cells and primary human trophoblasts (PHT cells) cultured in lipoprotein-deficient medium. We also measured the effect of the synthetic, LXR-specific ligand T0901317 on PHT cell differentiation and survival. Finally, we silenced the ATP-binding cassette transporter A1 (ABCA1), a transcriptional target of LXR that drives cholesterol efflux, to determine if inhibition of cholesterol efflux could block the effects of T0901317. ResultsOxysterols inhibited BeWo survival and PHT cell differentiation, and these effects were blocked by cholesterol supplementation. T0901317 also inhibited PHT cell differentiation, and this effect was similarly blocked by cholesterol. Unlike cholesterol however, ABCA1 silencing did not modify the effect of T0901317 on PHT cell differentiation. DiscussionOxysterols and LXR inhibit trophoblast survival and differentiation exclusively in conditions of cholesterol scarcity. These findings underscore the importance of cholesterol homeostasis in the maintenance of placental function and suggest that pathways regulating cholesterol homeostasis may represent therapeutic targets to mitigate harmful sequelae of placental injury.

Influence of Cooking, Storage Period, and Re-heating on Production of Cholesterol Oxides in Chicken Meat.

The objective of present study was to investigate the effect of cooking and their combinations with re-heating methods on the formation of cholesterol oxidation products (COPs) in stored chicken thigh meat. Pan roasting, steaming, oven grilling, charcoal grilling, and microwaving were used for cooking. Re-heating of samples was done using the same cooking methods or microwaving after 3 and 6 d of refrigerated storage. Cooking and re-heating resulted in reduction of crude fat and cholesterol contents of chicken thigh meat depending on storage period before re-heating. Cooking and storage period had no influence on the total amount of COPs. The highest total amount of COPs was observed in meat samples cooked by steaming and reheated by microwaving after 6 d of storage, which showed similar value to raw chicken meat stored for 6 days. However, different re-heating methods formed different types of COPs depending on storage period before re-heating. The high amount (p<0.05) of 25-hydroxycholesterol or α-epoxide was detected in meat samples reheated by steaming or microwaving at 3 or 6 d of storage after steamed cooking, respectively. As a result, the combination of steaming and re-heating with microwaving could increase the total amount of COPs in chicken thigh meat and different cooking/re-heating methods could form different types of COPs, even though no significant difference in the total amount of COPs depending on storage period.

Cholesterol oxidation products in kefir from goats’ milk during storage

Cholesterol oxidation products were identified in goats’ whole fat milk kefir when stored under different light sources (500–590 cd) and luminous flux (90–120 Lm). The total sum of cholesterol oxidation products was 1.75 mg kg−1 fat, which after 4 weeks increased to 6.85 mg kg−1 fat. The largest component among the identified oxysterols was 7β-hydroxycholesterol (0.72 mg kg−1 fat), and secondly 25-hydroxycholesterol (0.41 mg kg−1 fat). The storage time and light intensity influenced the formation of 7-ketocholesterol. After 4 weeks at a light intensity of 450 lx, the amount of 7-ketocholesterol in the kefir was 1.92 mg kg−1 fat, and at an intensity of 80 lx it was 0.98 mg kg−1 fat.

The role of autophagy in survival response induced by 27-hydroxycholesterol in human promonocytic cells

Autophagy has been shown to be stimulated in advanced atherosclerotic plaques by metabolic stress, inflammation and oxidized lipids. The lack of published studies addressing the potential stimulation of pro-survival autophagy by oxysterols, a family of cholesterol oxidation products, has prompted our study. Thus, the goal of the current study is to elucidate the molecular mechanism of the autophagy induced by 27-hydroxycholesterol (27-OH), that is one of the most abundant oxysterols in advanced atherosclerotic lesions, and to assess whether the pro-oxidant effect of the oxysterol is involved in the given response. Here we showed that 27-OH, in a low micromolar range, activates a pro-survival autophagic response in terms of increased LC3 II/LC3 I ratio and Beclin 1, that depends on the up-regulation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt pathways as a potential result of an intracellular reactive oxygen species increase provoked by the oxysterol in human promonocytic U937 cells. Moreover, 27-OH induced autophagy is dependent on the relation between nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-dependent antioxidant response and p62. The data obtained highlight the involvement of cholesterol oxidation products in the pathogenesis of oxidative stress related chronic diseases like atherosclerosis. Therefore, deeply understanding the complex mechanism and generating synthetic or natural molecules targeting this survival mechanism might be very promising tools in the prevention of such diseases.

A silver lining for 24-hydroxycholesterol in Alzheimer's disease: The involvement of the neuroprotective enzyme sirtuin 1

It is now established that cholesterol oxidation products (oxysterols) are involved in several events underlying Alzheimer's disease (AD) pathogenesis. Of note, certain oxysterols cause neuron dysfunction and degeneration but, recently, some of them have been shown also to have neuroprotective effects. The present study, which aimed to understand the potential effects of 24-hydroxycholesterol (24-OH) against the intraneuronal accumulation of hyperphosphorylated tau protein, stressed these latter effects. A beneficial effect of 24-OH was demonstrated in SK-N-BE neuroblastoma cells, and is due to its ability to modulate the deacetylase sirtuin 1 (SIRT1), which contributes to preventing the neurotoxic accumulation of the hyperphosphorylated tau protein. Unlike 24-OH, 7-ketocholesterol (7-K) did not modulate the SIRT1-dependent neuroprotective pathway. To confirm the neuroprotective role of 24-OH, in vivo experiments were run on mice that express human tau without spontaneously developing tau pathology (hTau mice), by means of the intracerebroventricular injection of 24-OH. 24-OH, unlike 7-K, was found to completely prevent the hyperphosphorylation of tau induced by amyloid β monomers. These data highlight the importance of preventing the loss of 24-OH in the brain, and of maintaining high levels of the enzyme SIRT1, in order to counteract neurodegeneration. Graphical abstractA hypothetical scheme of the molecular mechanisms underlying the effects of 24-OH on hyperphosphorylated tau accumulation. [Display omitted]

Lipid Oxidation Derived Aldehydes and Oxysterols Between Health and Disease

Non‐enzymatic and enzymatic oxidation of lipids has long been a primary issue in human pathophysiology, with special emphasis on diet‐ and nutrition‐related aspects. While there is no doubt that the n‐3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid, and docosahexaenoic acid, exert anti‐inflammatory, inflammation resolving, and immunomodulatory effects in different model systems, their non‐enzymatic oxidation during food storage and cooking, as well as during gastrointestinal digestion, can give rise to excessive amounts of quite reactive aldehydic end‐products, in particular 4‐hydroxyhexenal. However, the non‐enzymatic oxidation of n‐6 PUFAs is, in principle, far more harmful, not least because one of its main end‐products, 4‐hydroxynonenal, has been shown to be associated with the progression of a number of human diseases. Several cholesterol oxidation products, known as oxysterols, are also of primary nutritional importance; these can be generated in the human body through both enzymatic and non‐enzymatic reactions, but may also be present in the diet in excessive amounts due to the autoxidation of cholesterol‐containing foods. Their potential contribution to the pathogenesis and progression of human diseases in which hypercholesterolemia is a primary risk factor, such as atheroclerosis, Alzheimer's disease, and some forms of cancers, has been unanimously recognized.Practical Applications: This review highlights the importance of certain lipid oxidation products, namely 4‐hydroxyalkenals and oxysterols, in the modulation of processes fundamental to human health and disease. The observations and facts reviewed here underline the need to carefully consider the pros and cons of the dietary intake and metabolic fate of n‐3 PUFA, n‐6 PUFA, and cholesterol, when producing, storing, and processing lipid‐containing food.Lipid oxidation is a common process that occurs through both enzymatic and non‐enzymatic reactions. It is responsible for the formation of several compounds, including polyunsaturated fatty acid (PUFA)‐derived 4‐hydroxyalkenals and cholesterol oxidation products, namely oxysterols. Depending on their concentrations, these molecules possess physiopathological properties involved in cell viability and function. At high concentrations, such as those resulting from intense oxidative stress or excessive fatty food intake, they are associated to the onset of many diseases. A correct diet regimen, improved food processing and storage, and suitable antioxidant supplementation, should be implemented to counteract promotion of disease development by excess oxidized lipids. (HHE, 4‐hydroxyhexenal; HNE, 4‐hydroxynonenal; ROS, reactive oxygen species).

Montmorency tart cherry anthocyanins: dose‐dependent antioxidant activity against cholesterol oxidation

Cardiovascular disease (CVD) is a leading cause of death world‐wide, and cholesterol oxidation (CO) and cholesterol oxidation products (COPs) are widely thought to contribute to CVD by exacerbating mechanisms that produce atherosclerotic plaques. COPs not only hold atherogenic properties, but also mutagenic, carcinogenic and inflammatory properties. More recently, it has been reported that they also play an important role in the onset and progression of neurodegenerative diseases. As promoters of inflammation, cytotoxicity, and fibrogenesis, COPs drive a cycle of inflammation that increases the steady‐state level of radical and non‐radical reactive oxygen species (ROS), which in turn amplify inflammation.Strong evidence indicates that natural products derived from plants, such as anthocyanins, poses antioxidant properties and may inhibit lipid oxidation and its metabolites. Montmorency cherries (Prunus cerasus) contain polyphenols in amounts of 3000 mg/100g dry weight, because of this they could act as a natural source of strong antioxidants. Our group has fractionated and characterized different polyphenols from Montmorency tart cherry extracts. Our preliminary studies indicate that anthocyanins have potent dose‐dependent antioxidants activity against CO suggesting that they may also be efficient at reducing CO‐induced inflammation in the CVD. We explore the antioxidant properties of Montmorency tart cherry anthocyanins against CO using in vitro systems of CVD, such as biomimetic liposomes and low‐density lipoprotein (LDL). Radical‐induced oxidation was triggered by addition of AAPH radical generator at 0.5–2 mM to a large unilamellar vesicles (LUVs) suspension under physiological conditions. Different concentrations of anthocyanins (2–100 μM) were tested over 180 minutes.Results demonstrated that anthocyanins may have a dose‐dependent antioxidant activity, specifically targeting CO. UV‐Vis spectroscopy showed that an increase in the oxidation of liposomal cholesterol when AAPH was added to the system (Control). A dramatic increase in cholesterol oxidation was observed between 180 and 240 minutes. The kinetics associated with the formation of cholesterol hydroperoxides were delayed up 180 min, with subsequent decrease on the accumulation of 7‐ketocholesterol (7‐Keto) and 25‐hydroxycholesterol (25‐OH), which are biomarkers of CO and thus the cardiovascular inflammation. We speculate that specific compounds derived from Montmorency tart cherries have the ability of to penetrate lipid membrane and scavenge ROS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elevation of plasma lysosphingomyelin-509 and urinary bile acid metabolite in Niemann-Pick disease type C-affected individuals

Niemann-Pick disease type C (NPC) is a neurovisceral disorder associated with the accumulation of lipids such as cholesterol and sphingolipids. NPC is caused by either NPC1 or NPC2, which encode lysosomal proteins located at membraneous and soluble fractions, respectively. For the past decade, the oxidation products of cholesterol, such as cholestane-3β,5α,6β-triol and 7-ketocholesterol, have been considered selective biomarkers for NPC. However, recent evidence has indicated numerous novel biomarkers for NPC, which raises the possibility that the diagnosis of NPC might be associated with the elevation of multiple lipid biomarkers, rather than a single biomarker. Sphingosylphosphorylcholine (SPC) has been suggested to be one such biomarker for NPC, in which elevated sphingomyelin is a potential precursor. Thus, we first performed a validation study of plasma SPC using LC-MS/MS. The results showed the following plasma concentrations in the NPC-affected and control individuals, respectively: 8.2 ± 2.8 nM (mean ± SD; median, 7.0 nM; max, 11.7 nM; min, 5.1 nM; n = 5) and 3.1 ± 1.4 nM (median, 2.9 nM; max, 4.8 nM; min, 1.5 nM; n = 7). We further extended the study to plasma lysophingomyelin-509 for NPC, a newly reported biomarker with uncharacterized chemical nature. Based on these result with plasma SPC as a surrogate marker, the value of mean of median of plasma lysophingomyelin-509 in NPC-affected individuals elevated at 65.2 (max, 73.2; min, 26.7; n = 5). Furthermore, the efficacy of plasma SPC and lysosphingomyelin-509 as promising biomarkers for this disorder was supported by the finding that the urinary concentration of 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid, an established biomarker for NPC, was also elevated in the NPC-affected individuals. These results suggest that a novel combination of plasma biomarkers, such as SPC and/or lysophingomyelin-509, and urinary bile acid metabolite could offer a promising platform for the diagnosis of NPC.

Short communication: Cholesterol oxidation products in traditional buttermilk

The aim of this study was to quantitatively and qualitatively assess the content of cholesterol oxidation products in traditional buttermilk after butter production. Cholesterol oxidation products (COP) exhibit a wide spectrum of biological activity, including cytotoxic, carcinogenic, and pro-oxidative properties. Buttermilk has about 2 mg of COP/kg of fat, including 7β-hydroxycholesterol and 5,6α-epoxycholesterol. Buttermilk immediately after production had a relatively high level of 7β-hydroxycholesterol (1.47 mg/kg), which decreased to 0.61 mg/kg after storage for 10 h at 3°C. During storage, the content of 5,6α-epoxycholesterol increased from 0.50 to 1.40 mg/kg. After 10 h of storage, the antioxidant potential of the buttermilk decreased (expressed as radical scavenging ability; change in 1,1-diphenyl-2-picrylhydrazyl = 32.2%). This study showed the presence of COP in fresh and stored buttermilk and the influence of time on changing the direction of cholesterol oxidation.

Lipid Biomarkers for the Peroxisomal and Lysosomal Disorders: Their Formation, Metabolism and Measurement

Lipid biomarkers play important roles in the diagnosis of and monitoring of treatment in peroxisomal disorders and lysosomal storage disorders. Today, a variety of lipids, including very long chain fatty acids, glycolipids, bile acids and the oxidation products of cholesterol, have been considered as biomarkers for these disorders. In this brief review, the authors summarized the recent advances regarding these lipid biomarkers in terms of their formation, metabolism and measurement in these disorders. An understanding of these biomarkers will offer a key to the development of novel diagnoses and help create more effective therapies in the future.

Derangement of intestinal epithelial cell monolayer by dietary cholesterol oxidation products

The emerging role of the diet in the incidence of intestinal inflammatory diseases has stimulated research on the influence of eating habits with pro-inflammatory properties in inducing epithelial barrier disturbance. Cholesterol oxidation products, namely oxysterols, have been shown to promote and sustain oxidative/inflammatory reactions in human digestive tract. This work investigated in an in vitro model the potential ability of a combination of dietary oxysterols representative of a hyper-cholesterol diet to induce the loss of intestinal epithelial layer integrity.The components of the experimental mixture were the main oxysterols stemming from heat-induced cholesterol auto-oxidation, namely 7-ketocholesterol, 5α,6α-and 5β,6β-epoxycholesterol, 7α- and 7β-hydroxycholesterol. These compounds added to monolayers of differentiated CaCo-2 cells in combination or singularly, caused a time-dependent induction of matrix metalloproteinases (MMP)-2 and -9, also known as gelatinases. The hyperactivation of MMP-2 and -9 was found to be associated with decreased levels of the tight junctions zonula occludens-1 (ZO-1), occludin and Junction Adhesion Molecule-A (JAM-A). Together with such a protein loss, particularly evident for ZO-1, a net perturbation of spatial localization of the three tight junctions was observed.Cell monolayer pre-treatment with the selective inhibitor of MMPs ARP100 or polyphenol (-)-epicathechin, previously shown to inhibit NADPH oxidase in the same model system, demonstrated that the decrease of the three tight junction proteins was mainly a consequence of MMPs induction, which was in turn dependent on the pro-oxidant property of the oxysterols investigated. Although further investigation on oxysterols intestinal layer damage mechanism is to be carried on, the consequent - but incomplete - prevention of oxysterols-dependent TJs alteration due to MMPs inhibition, avoided the loss of scaffold protein ZO-1, with possible significant recovery of intestinal monolayer integrity.

Olive Oil Phenolics Prevent Oxysterol-Induced Proinflammatory Cytokine Secretion and Reactive Oxygen Species Production in Human Peripheral Blood Mononuclear Cells, Through Modulation of p38 and JNK Pathways.

ScopeThe aim of the present study was to investigate the ability of extra virgin olive oil (EVOO) polyphenols to counteract the proinflammatory effects induced by dietary and endogenous oxysterols in ex vivo immune cells.Methods and resultsPeripheral blood mononuclear cells (PBMCs), separated from the whole blood of healthy donors, were utilized and were stimulated with an oxysterols mixture, in the presence of physiologically relevant concentrations of the EVOO polyphenols, hydroxytyrosol, tyrosol, and homovanillic alcohol. Oxysterols significantly increased the production of proinflammatory cytokines, interleukin‐1β, regulated on activation, normal T‐cell expressed and secreted and macrophage migration inhibitory factor in ex vivo cultured PBMCs. Increased levels of reactive oxygen species (ROS) were also detected along with increased phosphorylation of the p38 and JNK. All phenolic compounds significantly reduced cytokine secretion induced by the oxysterols and inhibited ROS production and mitogen activated protein kinase phosphorylation.ConclusionsThese results suggest that extra virgin olive oil polyphenols modulate the immune response induced by dietary and endogenous cholesterol oxidation products in human immune cells and may hold benefit in controlling chronic immune and/or inflammatory processes.

Dietary docosahexaenoic acid supplementation prevents the formation of cholesterol oxidation products in arteries from orchidectomized rats.

Testosterone deficiency has been correlated with increased cardiovascular diseases, which in turn has been associated with increased oxidative stress. Several studies have considered cholesterol oxidation products (COPs) as oxidative stress biomarkers, since some of them play pro-oxidant and pro-inflammatory roles. We have previously described the cardioprotective effects of a dosahexaenoic acid (DHA) supplemented diet on the aortic and mesenteric artery function of orchidectomized rats. The aim of this study was to investigate whether impaired gonadal function alters the formation of COPs, as well as the potential preventive role of a DHA-supplemented diet on that effect. For this purpose, aortic and mesenteric artery segments obtained from control and orchidectomized rats, fed with a standard or supplemented with DHA, were used. The content of the following COPs: 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 5,6α-epoxycholesterol, 5,6β-epoxycholesterol, cholestanetriol and 25-hydroxycholesterol, were analyzed by gas chromatography. The results showed that orchidectomy increased the formation of COPs in arteries from orchidectomized rats, which may participate in the orchidectomy-induced structural and functional vascular alterations already reported. The fact that the DHA-supplemented diet prevented the orchidectomy-induced COPs increase confirms the cardiovascular protective actions of DHA, which could be of special relevance in mesenteric arterial bed, since it importantly controls the systemic vascular resistance.

Oxysterols Versus Cholesterol in Model Neuronal Membrane. I. The Case of 7-Ketocholesterol. The Langmuir Monolayer Study

Oxysterols are products of cholesterol oxidation. They can be formed endogenously (in both enzymatic and non-enzymatic reactions) as well as exogenously (delivered with food). Recent studies clearly demonstrate cytotoxic properties of these compounds, being mainly due to their incorporation into natural lipid bilayers. This process can influence mechanical and physicochemical properties of biomembrane—mainly by modifying the interactions between its components, which may result in the disruption of proper functioning of cell membrane and could lead to its degradation. Therefore, it can be assumed that oxysterols may affect the initiation of neurodegenerative diseases, including Alzheimer’s disease. However, the mode of action of these molecules at the molecular level is not fully known. To get a better understanding of the role of oxysterols in neurodegeneration, it is of great importance to examine mutual interactions between oxysterols and neuronal membrane components. One of the most promising techniques that can be used to analyze such interactions is the Langmuir monolayer technique. In this work, we have prepared an artificial neuronal membrane modeled as multicomponent Langmuir monolayer built up with cholesterol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and sphingomyelin (SM). To examine whether there are any changes in the membrane properties under oxidative stress, in this paper we have investigated the impact of the representative ring-oxidized oxysterol: 7-ketocholesterol (7-KC). Our results show that replacing cholesterol with 7-KC increases the interaction between molecules in the model membrane.

Trace-level determination of eight cholesterol oxidation products in human plasma by dispersive liquid–liquid microextraction and ultra-performance liquid chromatography–tandem mass spectrometry

ABSTRACT7α-Hydroxy cholesterol (7α-OHC), 25-hydroxy cholesterol (25-OHC), 27-hydroxy cholesterol (27-OHC), 4β-hydroxy cholesterol (4β-OHC), 7α-hydroxy-4-cholesten-3-one (7α-C4), 5β-cholestane-3α, 7α, 12α-triol (5β-Triol), cholic acid (CA), and chenodeoxycholic acid (CDCA) are known biomarkers of neurodegenerative diseases. A method for their simultaneous determination in human plasma has been optimized using dispersive liquid–liquid microextraction and ultra-performance liquid chromatography–tandem mass spectrometry. The limits of quantification of the target compounds were in the range of 0.3–3.3 µg/L. The precision achieved by this method was less than 13.4% for intraday and interday analyses. The proposed method was used to analyze eight cholesterol oxidation products in 30 human plasma samples. The analytical results were in a concentration range of 1.6–87.4 µg/L for 7α-OHC, 6.3–58.2 µg/L for 25-OHC, 12.1–98.5 µg/L for 27-OHC, 5.7–64.8 µg/L for 4β-OHC, 1.5–124.1 µg/L for 7α-C4, 0.5–16.5 µg/L for 5β-Triol, 13.1–245 µg/L for CA, and 19.6–487 µg/L for CDCA in the samples. The method may be used for the analysis of biomarkers of neurodegenerative diseases.

7-ketocholesterol and 27-hydroxycholesterol decreased doxorubicin sensitivity in breast cancer cells: estrogenic activity and mTOR pathway.

Hypercholesterolemia is one of the risk factors for poor outcome in breast cancer therapy. To elucidate the influence of the main circulating oxysterols, cholesterol oxidation products, on the cell-killing effect of doxorubicin, cells were exposed to oxysterols at a subtoxic concentration. When cells were exposed to oxysterols in fetal bovine serum-supplemented medium, 7-ketocholesterol (7-KC), but not 27-hydroxycholesterol (27-HC), decreased the cytotoxicity of doxorubicin in MCF-7 (high estrogen receptor (ER)α/ERβ ratio) cells and the decreased cytotoxicity was restored by the P-glycoprotein inhibitor verapamil. 7-KC stimulated the efflux function of P-glycoprotein and reduced intracellular doxorubicin accumulation in MCF-7 but not in ERα(-) MDA-MB-231 and the resistant MCF-7/ADR cells. In MCF-7 cells, 7-KC increased the mRNA and protein levels of P-glycoprotein. The 7-KC-suppressed doxorubicin accumulation was restored by the fluvestrant and ERα knockdown. In a yeast reporter assay, the ERα activation by 7-KC was more potent than 27-HC. 7-KC, but not 27-HC, stimulated the expression of an ER target, Trefoil factor 1 in MCF-7 cells. When charcoal-stripped fetal bovine serum was used, both 7-KC and 27-HC induced Trefoil factor 1 expression and reduced doxorubicin accumulation in MCF-7 cells. 7-KC-reduced doxorubicin accumulation could be reversed by inhibitors of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin (mTOR). These findings demonstrate that 7-KC decreases the cytotoxicity of doxorubicin through the up-regulation of P-glycoprotein in an ERα- and mTOR-dependent pathway. The 7-KC- and 27-HC-elicited estrogenic effects are crucial in the P-glycoprotein induction in breast cancer cells.

Taxifolin reduces the cholesterol oxidation product-induced neuronal apoptosis by suppressing the Akt and NF-κB activation-mediated cell death.

The taxifolin effect on the cholesterol oxidation product-induced neuronal apoptosis was investigated using differentiated PC12 cells and human neuroblastoma SH-SY5Y cells. 7-ketocholesterol induced phosphorylation of Akt, and increase in the levels of cytosolic and nuclear NF-κB p65, cytosolic NF-κB p50 and cytosolic phosphorylated-IκB-α in PC12 cells. The cholesterol oxidation products also induced a decrease in the levels of Bid and Bcl-2, increase in the levels of p53 and Bax, loss of the mitochondrial transmembrane potential, release of cytochrome c, activation of caspases (-8, -9 and -3), production of reactive oxygen species, depletion of GSH and cell death in both cell lines. Taxifolin, N-acetylcysteine, trolox, Akt inhibitor and Bay11-7085 attenuated the cholesterol oxidation product-induced changes in the apoptosis-related protein levels, activation of the Akt and NF-κB, reactive oxygen species production, GSH depletion and cell death. These results show that taxifolin may reduce the cholesterol oxidation product-induced neuronal apoptosis by suppressing the Akt and NF-κB activation-mediated cell death. The suppressive effect appears to be attributed to the inhibition of reactive oxygen species production and GSH depletion.

Enzyme activities of α-glucosidase in Japanese neonates with pseudodeficiency alleles

Lysosomal storage disorders (LSDs) are caused by defective enzyme activities in lysosomes, characterized by the accumulation of sphingolipids, glycolipids, oligosaccharides, mucopolysaccharides, the oxidation products of cholesterol, and other biological substances. A growing number of clinical studies have suggested the enhanced efficacy of existing therapies, including enzyme replacement therapy, which is effective when it is initiated during the presymptomatic period. Thus, the identification of disease-affected individuals by newborn screening has been considered an effective platform. Previous studies have suggested that the discrimination of infantile-onset Pompe disease (IOPD) requires multi-step examination of GAA enzyme activity using the fluorometric technique. In sharp contrast, the MS/MS-based technique can identify the population of IOPD and the pseudodeficiency alleles of the GAA enzyme [Liao HC et al. Clin Chem (2017) in press; doi: http://dx.doi.org/10.1373/clinchem.2016.269027]. To determine whether MS/MS-based assay can identify these two populations in Japanese neonates, we first performed a validation study of this assay using flow-injection analysis (FIA)-MS/MS and liquid chromatography (LC)-MS/MS followed by examination of GAA enzyme activity in our population. By minimizing the effect of substrate-derived in-source decomposition products, the activities of 6 LSD enzymes were quantified in FIA-MS/MS and LC-MS/MS. The mean value of GAA activity with IOPD, pseudodeficiency alleles, and healthy controls by FIA-MS/MS were 1.0±0.3μmol/h/L (max, 1.3; min, 0.7; median, 1.2; n=3), 2.7±0.7μmol/h/L (max, 4.5; min, 1.5; median, 2.5; n=19), and 12.9±5.4μmol/h/L (max, 29.6; min, 2.5; median, 11.0; n=83), respectively. These results suggest that the population of GAA with pseudodeficiency alleles has approximately 20% of GAA enzyme activity compared to controls, providing the preliminary evidence to estimate the cut-off values in the Japanese population using this technique.

Effect of phenols extracted from a by-product of the oil mill on the shelf-life of raw and cooked fresh pork sausages in the absence of chemical additives

Replacing chemical additives in meat preparations with natural compounds is a matter of great interest today, both to consumers and to the food sector. The effect of an extract rich in phenols obtained from olive vegetation water (an agricultural by-product) on the pH, weight loss after cooking, diacylglycerols (DAGs), peroxide value (POV), thiobarbituric acid reactive species (TBARS), and cholesterol oxidation products (COPs) of raw and cooked fresh pork sausages prepared without chemical additives was evaluated before and after aerobic storage at 2 ± 2 °C for 14 d. Adding the extract at concentrations of 0.075 and 0.15 g/100 g resulted in a decrease in pH, DAGs, POV, TBARS and COPs; notably, the COPs levels were 4- and 17-fold lower in raw and cooked sausages, respectively. Sensory analysis revealed significant differences between control samples and those enriched with the extract, but the enriched samples were never considered unpleasant by the panellists. Storing the raw sausages for 14 d and subsequently cooking them led to 58% and 49% decreases in phenols, respectively. The purified phenols from olive oil wastewater proved to be an effective antioxidant, thus demonstrating themselves to be a potential ingredient to ensure the quality and safety of meat preparations.