Cholesterol Oxidation Products Research Articles

S3-2 - Modulation of cell signaling pathways by oxysterols in age-related human diseases

Cholesterol oxidation products, named oxysterols, may derive from the diet or originate endogenously by autoxidative nonenzymatic modification of cholesterol as well as through enxymatic pathways involved in lipid metabolism and maintenance of cholesterol homeostasis. Oxysterols have been shown to exert several in vitro and in vivo biochemical activities of both physiologic and pathologic relevance and they appear to be implicated in the pathogenesis of various age-related chronic diseases, including atherosclerosis and Alzheimer's disease (AD), where hypercholesterolemia represents a primary risk factor, and a redox state impairment and inflammation seem to play a central role. Our recent studies show that, in cells of the macrophage lineage or in human neuronal cells (differentiated or not), respectively in the contest of atherosclerosis or AD, oxysterols can initiate specific signal transduction pathways that are relevant to the development of these diseases. Regarding atherosclerosis, we have observed that oxysterols can contribute to plaque instability and rupture by enhancing inflammatory responses and matrix turnover through an unbalanced up-regulation of MMP-9. Concerning AD, we have demonstrated that oxysterols may promote neuroinflammatory changes and accelerate APP processing toward β-amyloid production by up-regulating APP and BACE1 protein levels. In addition, TLR4, a key player of immune and inflammatory signaling responses, seems to have an important role in the pathogenesis of both atherosclerosis and AD.

The influence of ligand-activated LXR on primary human trophoblasts

IntroductionThe Liver X Receptors (LXRs) are critical transcriptional regulators of cellular metabolism that promote cholesterol efflux and lipogenesis in response to excess intracellular cholesterol. In contrast, the Sterol Response Element Binding Protein-2 (SREBP2) promotes the synthesis and uptake of cholesterol. Oxysterols are products of cholesterol oxidation that accumulate in conditions associated with increased cellular levels of reactive oxygen species, such as hypoxia and oxidative stress, activating LXR and inhibiting SREBP2. While hypoxia and oxidative stress are commonly implicated in placental injury, the impact of the transcriptional regulation of cholesterol homeostasis on placental function is not well characterized. MethodsWe measured the effects of the synthetic LXR ligand T0901317 and the endogenous oxysterol 25-hydroxycholesterol (25OHC) on differentiation, cytotoxicity, progesterone synthesis, lipid droplet formation, and gene expression in primary human trophoblasts. ResultsExposure to T0901317 promoted lipid droplet formation and inhibited differentiation, while 25OHC induced trophoblast toxicity, promoted hCG and progesterone release at lower concentrations with inhibition at higher concentrations, and had no effect on lipid droplet formation. The discrepant effect of these ligands was associated with distinct changes in expression of LXR and SREBP2 target genes, with upregulation of ABCA1 following 25OHC and T090317 exposure, exclusive activation of the lipogenic LXR targets SREBP1c, ACC1 and FAS by T0901317, and exclusive inhibition of the SREBP2 targets LDLR and HMGCR by 25OHC. ConclusionThese findings implicate cholesterol oxidation as a determinant of trophoblast function and activity, and suggest that placental gene targets and functional pathways are selectively regulated by specific LXR ligands.

Cholesterol transformations during heat treatment

The aim of the study was to characterise products of cholesterol standard changes during thermal processing. Cholesterol was heated at 120°C, 150°C, 180°C and 220°C from 30 to 180min. The highest losses of cholesterol content were found during thermal processing at 220°C, whereas the highest content of cholesterol oxidation products was observed at temperature of 150°C. The production of volatile compounds was stimulated by the increase of temperature. Treatment of cholesterol at higher temperatures i.e. 180°C and 220°C led to the formation of polymers and other products e.g. cholestadienes and fragmented cholesterol molecules. Further studies are required to identify the structure of cholesterol oligomers and to establish volatile compounds, which are markers of cholesterol transformations, mainly oxidation.

Structural characterization of human cholesterol 7α-hydroxylase

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B' helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.

Niemann-Pick disease type C: introduction and main clinical features.

Niemann-Pick disease type C: introduction and main clinical features.

Effect of dietary carnosic acid on meat quality from suckling lambs

In order to elucidate the influence of dietary carnosic acid on the quality of suckling-lamb meat, twenty-four lambs were fed ad libitum daily with milk replacer (MR) alone (control group, CTRL), enriched with carnosic acid (CARN, 0.096gkg−1 live weight, LW), or vitamin E (VITE, 0.024gkg−1, LW), the last group being considered as a positive control. Animals were slaughtered at the intended body weight (11–12kgLW). Longissimus thoracis muscles were used to asses proximate composition of meat, whereas different muscles (longissimus lumborum and gluteus medius) were sliced and kept refrigerated during 0, 7, and 14 days to determine water holding capacity, thiobarbituric acid reactive substances (TBARS), and cholesterol oxidation products (COPs) in cooked meat samples. Biceps femoris muscles were used for the analysis of volatile compounds on precooked meat after 1 and 7 days of storage. The results indicate that, at the dose used, carnosic acid dietary supplementation seemed to be less effective than vitamin E reducing lipid oxidation of suckling-lambs meat.

Oxidative stability of n-3-enriched chicken patties under different package-atmosphere conditions

The oxidation processes were studied in chicken patties, enriched with n-3 fatty acids, after 8days of storage at 4°C, under different aerobic conditions, and following heat treatment. Significant effects were seen on lipid and cholesterol oxidation and the sensory qualities for whole flaxseed addition in the chicken feed (i.e., n-3 fatty acid enrichment), and for the different package-atmosphere conditions. For the raw chicken patties, n-3 enrichment increased the colour L∗ values while, after the heat treatment, there were higher thiobarbituric acid-reactive substances (TBARs) and cholesterol oxidation products (COPs), and the rancidity was more pronounced. In comparison with the low O2 (<0.5%) package-atmosphere condition, O2 enrichment (80%) increased the instrumentally measured colour values, TBARs, total and individual COPs, and the rancidity became pronounced. The most suitable package-atmosphere condition of these raw n-3-enriched chicken patties is a very low O2 atmosphere, with or without an O2 scavenger.

Do n-3 polyunsaturated fatty acids increase or decrease lipid peroxidation in humans?

Abstract Background: Despite many known health benefits of n-3 polyunsaturated fatty acids (PUFA), there is a concern that their high degree of unsaturation may actually increase oxidative stress, lipid peroxidation (LPO), and chronic inflammatory diseases. In this review, we have analyzed results from published human studies regarding the effects of n-3 PUFA supplementation on markers of lipid peroxidation. Of the 22 published human studies, nine found no change, eight a decrease, and five an increase in markers of LPO. These inconsistencies may be due to methods, subject characteristics, dose, duration, fatty acid and antioxidant composition of supplements, and basal diets. METHODS used for analysis seem to be the most significant factor. Six of eight studies with a decrease in LPO determined F2-isoprostanes produced in vivo, and two determined plasma antioxidant capacity or hydroperoxides. n-3 PUFA can serve as scavengers for free radicals and also modulate expression of genes that determine the balance between oxidative and antioxidative status. Recent studies that monitored oxidation products of cholesterol and fatty acids support the hypothesis that n-3 PUFA decrease LPO. Most of the studies showing no change or increase in LPO determined markers that involved ex vivo sample preparation or oxidation (malondialdehyde, low-density lipoprotein oxidation, lipid hydroperoxides). A majority of studies do not indicate that n-3 PUFA increased LPO. Future studies need to investigate the effects of dose, duration, and composition of n-3 PUFA with standardized diets and methods on concentrations and types of LPO products produced.

Inhibitory activities of natural extracts on cholesterol oxidation products formation in beef patties

Inhibitory activities of natural extracts on cholesterol oxidation products formation in beef patties

In Vitro Effects of Cooking Methods on Digestibility of Lipids and Formation of Cholesterol Oxidation Products in Pork.

This study investigated the effects of cooking methods on the digestibility of lipids and formation of cholesterol oxidation products (COPs) in pork, during in vitro human digestion. Pork patties were cooked using four different methods (oven cooking, pan frying, boiling, and microwaving), to an internal temperature of approximately 85℃. The digestibility of pork patties were then evaluated, using the in vitro human digestion model that simulated the composition (pH, minerals, surfaceactive components, and enzymes) of digestive juices in the human mouth, stomach, and small intestine. The total lipid digestibility was higher after microwave cooking, whereas pan-frying resulted in lower in vitro digestibility, compared to the other cooking methods. The microwaving method followed by in vitro digestion also showed significantly higher content of free fatty acids and thiobarbituric acid reactive substances (TBARS), compared to the other cooking methods; whereas, the pan frying and boiling methods showed the lowest. Cholesterol content was not significantly different among the cooked samples before, and after in vitro human digestion. The formation of COPs was significantly higher in the microwave-treated pork samples, compared to those cooked by the other methods, which was consistent with the trend for lipid peroxidation (TBARS). We propose that from the point of view of COPs formation and lipid oxidation, the pan-frying or boiling methods would be useful.

Squalene oxidation products: Monitoring the formation, characterisation and pro‐oxidant activity

Squalene (SQ), a precursor of sterols and terpenoids is a functional lipid of high importance in the food and pharmaceutical sectors. SQ oxidation studies are rather limited compared with those for other olefins. The aim of the present study was to monitor the formation of SQ oxidation products under different conditions (temperature, air supply), to characterise the most abundant of them by spectroscopic techniques and then examine their pro‐oxidant activity in a model lipid substrate. Squalane (SQA), the saturated analogue of SQ, was used as a reference compound. FT‐MIR analysis indicated the presence of alcohols, epoxides, aldehydes, and ketones. GC‐MS was used to characterise SQ primary oxidation and scission products. The presence of epoxides was further confirmed by means of 1H NMR and 13C NMR spectroscopy. It could be argued that SQ stability is due to its stereochemistry and specifically to the presence of methyl groups next to the double bonds. The pro‐oxidant activity of SQ oxidation products was evident at 62 and 40°C and suppressed only in the presence of primary antioxidants, not of SQ. The present work adds to the characterisation of SQ oxidised products. To our knowledge their pro‐oxidant activity has never been examined before.Practical applications: Characterisation of squalene oxidation products and assessment of their activity as pro‐oxidants present both scientific interest regarding the kinetics and product identity as well as a practical impact in case this bioactive lipid is provided for consumption as a functional product. In the past, cholesterol oxidation products and more recently phytosterol ones attracted the interest of researchers, who studied the stability of the respective parent compounds for food safety reasons.Monitoring of the formation of SQ oxidation products under different conditions (temperature, air supply) and chemical characterisation of the most abundant of them by spectroscopic techniques. Examination of their pro‐oxidant activity in a model lipid substrate. Squalene may exert a weak antioxidant activity due to competitive oxidation phenomena with the lipid substrate while its oxidation products have a pro‐oxidant activity on purified olive oil model substrate that was suppressed only in the presence of primary antioxidants.

Effect of biopolymer encapsulation on the digestibility of lipid and cholesterol oxidation products in beef during in vitro human digestion

In this study, beef patties were encapsulated with 3% chitosan, pectin, onion powder, or green tea powder and the beef patties were then passed through an in vitro human digestion model. The total lipid digestibility was lowest (p<0.05) in beef patties encapsulated with chitosan and pectin after digestion in the small intestine. Thiobarbituric acid reactive substance (TBARS) values were significantly lower (p<0.05) for beef patties encapsulated with chitosan and pectin, when compared with the control, after digestion in the small intestine. In contrast, the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical–scavenging activity was highest (p<0.05) in beef patties encapsulated with onion powder and green tea powder after digestion in the small intestine. The total cholesterol oxidation product (COP) content was significantly lower (p<0.05) in beef patties encapsulated with biopolymers than in the control after digestion in the small intestine.

Loading into nanoparticles improves quercetin's efficacy in preventing neuroinflammation induced by oxysterols.

Chronic inflammatory events appear to play a fundamental role in Alzheimer's disease (AD)-related neuropathological changes, and to result in neuronal dysfunction and death. The inflammatory responses observed in the AD brain include activation and proliferation of glial cells, together with up-regulation of inflammatory mediators and of free radicals. Along with glial cells, neurons themselves can also react and contribute to neuroinflammatory changes in the AD brain, by serving as sources of inflammatory mediators. Because excess cholesterol cannot be degraded in the brain, it must be excreted from that organ as cholesterol oxidation products (oxysterols), in order to prevent its accumulation. Among risk factors for this neurodegenerative disease, a mechanistic link between altered cholesterol metabolism and AD has been suggested; oxysterols appear to be the missing linkers between the two, because of their neurotoxic effects. This study shows that 24-hydroxycholesterol, 27-hydroxycholesterol, and 7β-hydroxycholesterol, the three oxysterols potentially implicated in AD pathogenesis, induce some pro-inflammatory mediator expression in human neuroblastoma SH-SY5Y cells, via Toll-like receptor-4/cyclooxygenase-2/membrane bound prostaglandin E synthase (TLR4/COX-2/mPGES-1); this clearly indicates that oxysterols may promote neuroinflammatory changes in AD. To confirm this evidence, cells were incubated with the anti-inflammatory flavonoid quercetin; remarkably, its anti-inflammatory effects in SH-SY5Y cells were enhanced when it was loaded into β-cyclodextrin-dodecylcarbonate nanoparticles, versus cells pretreated with free quercetin. The goal of loading quercetin into nanoparticles was to improve its permeation across the blood-brain barrier into the brain, and its bioavailability to reach target cells. The findings show that this drug delivery system might be a new therapeutic strategy for preventing or reducing AD progression.

Effect of cooking conditions on cholesterol oxidation and astaxanthin in dried salted shrimp

Dried salted shrimp is a product made from raw shrimps, which are usually cooked and dried under direct sunlight. Brine cooking is an important step during the production of dried salted shrimp as it promotes changes that affect the product's end quality. The aim of this study was to evaluate the effect of brine concentration and boiling time on cholesterol oxidation products (COPs) formation and the concomitant changes in astaxanthin content and fatty acid profile in shrimp during cooking, sun drying, and storage. Boiling conditions did not affect COPs formation in shrimp after cooking. However, increased brine concentration and boiling time promoted high astaxanthin retention in cooked shrimp. During the first 24 h of sun drying, COPs formation in dried salted shrimp was influenced by the interaction between brine concentration and boiling time; in fact, the lowest COPs levels were observed in samples boiled at low brine concentration and short cooking times, as well as those boiled in brine with high salt concentration and long cooking times. Most astaxanthin (ca. 78%) present in cooked shrimp was degraded during solar drying. During storage, PUFA decreased, and a concomitant astaxanthin degradation and COPs formation in dried salted shrimp were observed. Neither boiling time nor storage at dark of dried salted shrimp prevented these changes.Practical applications: The content of oxysterols in cooked dried shrimps can be minimized by optimizing the processing conditions to minimize the oxidation. This is useful for processors and consumers to reduce the intake of these toxic forms of cholesterol.COPs and astaxanthin in dried shrimp were analysed during cooking, solar drying and storage. The COPs content in dried shrimp showed a dramatic increase after solar drying. During the first 24 h of sun drying, COPs formation in dried salted shrimp was influenced by the brine concentration and boiling time.

Stimulation of bone regeneration following the controlled release of water-insoluble oxysterol from biodegradable hydrogel

Recently bone graft substitutes using bone morphogenetic proteins (BMPs) have been heralded as potential alternatives to traditional bone reconstruction procedures. BMP-based products, however, are associated with significant and potentially life-threatening side effects when used in the head and neck region and furthermore, are exorbitantly priced. Oxysterols, products of cholesterol oxidation, represent a class of molecules that are favorable alternatives or adjuncts to BMP therapy due to their low side effect profile and cost. In order to establish the optimal clinical utility of oxysterol, an optimal scaffold must be developed, one that allows the release of oxysterol in a sustained and efficient manner. In this study, we prepare a clinically applicable bone graft substitute engineered for the optimal release of oxysterol. We first solubilized oxysterol in water by making use of polymeric micelles using l-lactic acid oligomer (LAo) grafted gelatin. Then, the water-solubilized oxysterol was incorporated into a biodegradable hydrogel that was enzymatically degraded intracorporeally. In this manner, oxysterol could be released from the hydrogel in a degradation-driven manner. The water-solubilized oxysterol incorporated biodegradable hydrogel was implanted into rat calvarial defects and induced successful bone regeneration. The innovative significance of this study lies in the development of a bone graft substitute that couples the osteogenic activity of oxysterol with a scaffold designed for optimized oxysterol release kinetics, all of which lead to better repair of bone defects.

Fatty acid composition, oxidation status and volatile organic compounds in “Colonnata” lard from Large White or Cinta Senese pigs as affected by curing time

The aim of the present paper was to evaluate the fatty acid composition, lipolysis, lipid oxidation and volatile organic compounds (VOCs) in Colonnata lard from Large White (LW) or Cinta Senese (CS) pigs during one-year of curing. CS lard contained higher amounts of unsaturated fatty acids than that from LW, due to the different rearing and feeding systems. Despite higher lipolysis in CS backfat during the curing period, the rate of fatty acid and cholesterol oxidation was higher in LW. The amount of cholesterol oxidation products (COPs) and thiobarbituric acid reactive substances (TBARs) significantly decreased after 3months of curing, regardless of the type of lard. VOCs composition of lard was affected by curing time, but not by breed. While volatile fatty acid oxidation products (mainly aldehydes) were present at the beginning of curing, subsequently other volatile compounds (such as sulphur compounds and terpenes) that derived from ingredients used for lard production, increased in the samples.

Formation of cholesterol oxidation products (COPs) and loss of cholesterol in fresh egg pasta as a function of thermal treatment processing

The aim of this paper was the quantification of cholesterol loss and of its oxidation products in fresh egg pasta submitted to different thermal treatments. COPs were analysed by LC-MS/MS. The results showed that the COP concentration increased with increasing heating severity (expressed as F 100/10 value) from 66.44μg/g in the raw pasta up to 117.9μg/g after 120min at the reference temperature of 100°C; under the same processing conditions, the cholesterol loss reached 75%. This study revealed that 7-ketocholesterol (7-keto) is the main COP found and showed a linear correlation between its concentration and (i) total COP amount (r2=0.981; p<0.05), (ii) TBARS value (r2=0.969; p<0.05) and (iii) heating time expressed as a logarithm of F 100/10 value (r2=0.999 e p<0.01). The total amount of COPs found in the product was very low when compared to literature values reported for dried egg pasta and did not exceed 0.32mg/kg.

Effects of isoflavone supplementation on disturbances in lipid metabolism and antioxidant system due to exogenous cholesterol oxidation products in rats

Dietary cholesterol oxidation products (COP) cause disturbances in lipid metabolism and antioxidant system. Here we investigated if isoflavone supplementation ameliorated COP-induced disturbances in lipid metabolism and antioxidant system. Male Sprague–Dawley rats (4weeks of age) were fed diets containing 0.2% COP supplemented with or without 0.15% isoflavone aglycones for 3weeks. Isoflavone supplementation significantly ameliorated the dietary COP-induced decrease in hepatic cholesterol 7α-hydroxylase activity. Isoflavone supplementation also promoted faecal bile acid excretion, restored the decrease in plasma triacylglycerol levels in COP-fed rats, and suppressed COP-induced oxidative stress. Thus, isoflavone supplementation may ameliorate COP-induced deleterious effects by activating hepatic cholesterol 7α-hydroxylase and decreasing COP levels in the body.

Effect of Flaxseed Meals and Extracts on Lipid Stability in a Stored Meat Product.

Flaxseeds have been recently in focus due to the antioxidant capacity of some of their compounds. However, there is a lack of easily accessible information concerning their activity against lipid oxidation in food systems. Therefore, the aim of the study was to determine the effect of defatted meals (DFM) and the aqueous extracts (AFE) obtained from brown and golden flaxseeds on lipid oxidation in pork meatballs. Fatty acid composition, peroxide value (PV), thiobarbituric acid reactive substances (TBARS) and cholesterol content were monitored during 6 months of freezer storage. Cholesterol oxidation products were identified and quantified. Both DFM and AFE limited fatty acid and cholesterol oxidation during meatball storage. Their antioxidant effect depended on flax variety (brown or golden) and preparation type (DFM or AFE). Lower level of PV and TBARS, compared with the ones with AFE, were noted in meatballs with DFM. Both DFM and AFE, from the brown seed variety, protect the lipids against oxidation to a higher extent. During the storage, a cholesterol degradation was observed. AFE (particularly from the brown variety) limited changes in cholesterol content. Moreover, they stabilized fatty acid composition of stored meatballs. However, DFM efficiently inhibited cholesterol oxidation.

Up‐regulation of β‐amyloidogenesis in neuron‐like human cells by both 24‐ and 27‐hydroxycholesterol: protective effect of N‐acetyl‐cysteine

An abnormal accumulation of cholesterol oxidation products in the brain of patients with Alzheimer’s disease (AD) would further link an impaired cholesterol metabolism in the pathogenesis of the disease. The first evidence stemming from the content of oxysterols in autopsy samples from AD and normal brains points to an increase in both 27-hydroxycholesterol (27-OH) and 24-hydroxycholesterol (24-OH) in the frontal cortex of AD brains, with a trend that appears related to the disease severity. The challenge of differentiated SK-N-BE human neuroblastoma cells with patho-physiologically relevant amounts of 27-OH and 24-OH showed that both oxysterols induce a net synthesis of Aβ1-42 by up-regulating expression levels of amyloid precursor protein and β-secretase, as well as the β-secretase activity. Interestingly, cell pretreatment with N-acetyl-cysteine (NAC) fully prevented the enhancement of β-amyloidogenesis induced by the two oxysterols. The reported findings link an impaired cholesterol oxidative metabolism to an excessive β-amyloidogenesis and point to NAC as an efficient inhibitor of oxysterols-induced Aβ toxic peptide accumulation in the brain.