7-Hydroxycholestrol as a possible biomarker of cellular lipid peroxidation: Difference between cellular and plasma lipid peroxidation

Polyunsaturated fatty acids and their esters are known to be susceptible to free radical-mediated oxidation, whereas cholesterol is thought to be more resistant to oxidation. In fact, it has been observed that in the case of plasma lipid peroxidation, the amount of oxidation products of polyunsaturated fatty acids such as linoleic acid was higher than that of cholesterol. In contrast, during oxidative stress-induced cellular lipid peroxidation, oxidation products of cholesterol such as 7-hydroxycholesterol (7-OHCh) were detected in greater amounts than those of linoleates such as hydroxyoctadecadienoic acid (HODE). There are several forms of oxidation products of cholesterol and linoleates in vivo, namely, hydroperoxides, as well as the hydroxides of both the free and ester forms of cholesterol and linoleates. To evaluate these oxidation products, a method used to determine the lipid oxidation products after reduction and saponification was developed. With this method, several forms of oxidation products of cholesterol and linoleates are measured as total 7-OHCh (t7-OHCh) and total HODE (tHODE), respectively. During free radical-mediated lipid peroxidation in plasma, the amount of tHODE was 6.3-fold higher than that of t7-OHCh. In contrast, when Jurkat cells were exposed to free radicals, the increased amount of cellular t7-OHCh was 5.7-fold higher than that of tHODE. Higher levels of t7-OHCh than those of tHODE have also been observed in selenium-deficient Jurkat cells and glutamate-treated neuronal cells. These results suggest that, in contrast to plasma oxidation, cellular cholesterol is more susceptible to oxidation than cellular linoleates. Collectively, cholesterol oxidation products at the 7-position may be a biomarker of cellular lipid peroxidation.     

Evidence of cell damage induced by major components of a diet-compatible mixture of oxysterols in human colon cancer CaCo-2 cell line

Cholesterol oxidation products, termed oxysterols, have been shown to be more reactive than unoxidized cholesterol, possessing marked pro-inflammatory and cytotoxic effects in a number of cells and tissues. Oxysterols, absorbed with the diet as products of cholesterol auto-oxidation, have recently been suggested to potentially interfere with homeostasis of the mucosal intestinal epithelium, by promoting and sustaining irreversible damage.     

Cholesterol oxidation products in the vascular remodeling due to atherosclerosis

Like the other oxidation products of the lipid moiety of plasma low density lipoproteins (LDL), cholesterol oxidation products are consistently found within the characteristic lesions of atherosclerosis, both in experimental animals and in man. A growing bulk of evidence suggests that oxysterols make a significant contribution to the vascular remodeling that occurs in atherosclerosis, being involved in various key steps of this complex process: endothelial cell dysfunction, adhesion of circulating blood cells, foam cell and fibrous cap formation, modulation of the extracellular matrix (ECM), vascular cell apoptosis and plaque’s instability. Moreover, oxysterols have been demonstrated to be at least one or two orders of magnitude more reactive than unoxidized cholesterol in exerting pro-inflammatory, pro-apoptotic, and pro-fibrogenic effects. Thus, a pathological level of cholesterol oxidation in the vasculature may be the missing molecular link between hypercholesterolemia and the formation of atherosclerotic lesions.     

Analysis of native and oxidized low-density lipoprotein oxysterols using gas chromatography—mass spectrometry with selective ion monitoring

SUMMARY

Cholesterol oxidation products have been demonstrated to possess a wide variety of biological properties and have been implicated in playing an important role in the development of atherosclerosis. We have developed an analytical method using capillary gas chromatography-mass spectrometry (GC-MS) for the analysis of cholesterol oxidation products in low-density lipoprotein (LDL). The method uses programmed multiple selected ion monitoring (SIM), providing enhanced sensitivity and accuracy of peak detection over full-scan mass spectra. The major oxidation products of cholesterol in oxidized LDL were identified as 7β-hydroxy-cholesterol and 7-keto-cholesterol. Minor products included 4β-hydroxy-cholesterol, 6β-hydroxy-cholesterol and cholesterol-5α,6α-epoxide. Native LDL contains 7-lathosterol, which is a biosynthetic precursor of cholesterol, as well as low levels of 7β-hydroxy-cholesterol and 7-keto-cholesterol. 7-Lathosterol was not detected in oxidized LDL. A time course oxidation of native LDL with 8 μM CuCl₂ demonstrated a rapid increase in 7β-hydroxy-cholesterol and 7-keto-cholesterol over the first 4 h. Cholesterol—5α,6α-epoxide, and β4-hydroxy- and 6β-hydroxy-cholesterol levels increased gradually, while 7-lathosterol decreased over the same period. This method was used to measure the levels of 7-lathosterol and cholesterol oxides in the LDL of 20 healthy subjects in order to establish the mean concentration and a reference range. This method can be used for the characterization and quantitation of oxysterols in native and oxidized LDL and may afford an additional index of oxidative modification of plasma lipoproteins.     

Concentrations of iron correlate with the extent of protein, but not lipid, oxidation in advanced human atherosclerotic lesions

Previous studies have provided compelling evidence for the presence of oxidized proteins and lipids in advanced human atherosclerotic lesions. The catalyst responsible for such oxidation is unknown and controversial. We have previously provided evidence for elevated levels of iron in lesions. In this study we hypothesized that if iron ions catalyzed protein and lipid oxidation in the artery wall, then there should be a positive correlation between these parameters. Iron concentrations in ex vivo healthy human arteries and advanced carotid lesions were quantified by electron paramagnetic resonance spectroscopy. Four specific side-chain oxidation products of proteins, and the lipid oxidation products 7-ketocholesterol and cholesterol ester alcohols and hydroperoxides, were quantified by HPLC in the same samples used for the iron measurements. Parent amino acids, cholesterol, and cholesterol esters were also quantified. Statistically elevated levels of iron, cholesterol, cholesterol esters, 7-ketocholesterol, and cholesterol ester alcohols and hydroperoxides were detected in advanced lesions compared with healthy control tissue. Iron levels correlated positively and strongly with all four markers of protein oxidation, but not with either marker of lipid oxidation. These data support the hypothesis that elevated levels of iron contribute to the extent of protein, but not lipid, oxidation in advanced human lesions.     

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.     

Cholesterol oxidation: health hazard and the role of antioxidants in prevention

Cholesterol is a molecule with a double bond in its structure and is therefore susceptible to oxidation leading to the formation of oxysterols. These oxidation products are found in many commonly-consumed foods and are formed during their manufacture and/or processing. Concern about oxysterols consumption arises from the potential cytotoxic, mutagenic, atherogenic, and possibly carcinogenic effects of some oxysterols. Eggs and egg-derived products are the main dietary sources of oxysterols. Thermally-processed milk and milk-derived products are another source of oxysterols in our diet. Foods fried in vegetable/animal oil, such as meats and French-fried potatoes, are major sources of oxysterols in the Western diet. Efforts to prevent or to reduce cholesterol oxidation are directed to the use of antioxidants of either synthetic or natural origin. Antioxidants are not only able to inhibit triglyceride oxidation, some of them can also inhibit cholesterol oxidation. Among synthetic antioxidants 2,6-ditertiarybutyl-4-methylphenol (BHT), and tertiary butylhydroquinone (TBHQ) can efficiently inhibit the thermal-induced oxidation of cholesterol. Some natural antioxidants, such as alpha- and gamma-tocopherol, rosemary oleoresin extract, and the flavonoid quercetin, show strong inhibitory action against cholesterol oxidation.     

Lipid peroxidation: mechanisms, inhibition, and biological effects

In the last 50 years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipids are oxidized by three distinct mechanisms; enzymatic oxidation, non-enzymatic, free radical-mediated oxidation, and non-enzymatic, non-radical oxidation. Each oxidation mechanism yields specific products. The oxidation of linoleates and cholesterol is discussed in some detail. The relative susceptibilities of lipids to oxidation depend on the reaction milieu as well as their inherent structure. Lipid hydroperoxides are formed as the major primary products, however they are substrates for various enzymes and they also undergo various secondary reactions. Phospholipid hydroperoxides, for example, are reduced to the corresponding hydroxides by selenoproteins in vivo. Various kinds of antioxidants with different functions inhibit lipid peroxidation and the deleterious effects caused by the lipid peroxidation products. Furthermore, the biological role of lipid peroxidation products has recently received a great deal of attention, but its physiological significance must be demonstrated in future studies.     

Cholesterol photosensitized oxidation in food and biological systems

Lipid oxidation is one of the main chemical degradations occurring in biological systems and leads to the formation of compounds that are related to aging and various chronic and degenerative diseases. The extent of oxidation will depend on the presence of antioxidants/pro-oxidants, the unsaturation degree of fatty acids, and environmental conditions. Lipid oxidation can also affect other molecules that have double bonds in their chemical structures, such as cholesterol. Cholesterol oxidation products (COPs) have been studied in depth, because of their negative and controversial biological effects. The formation of COPs can be particularly favored in the presence of light and photosensitizers, since they generate excited singlet oxygen that rapidly reacts with the double bond by a non radical mechanism and without any induction period. The present review intends to provide an overall and critical picture of cholesterol photosensitized oxidation in food and biological systems, and its possible impact on human health and well-being.     

The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL

For more than two decades, there has been continuing evidence of lipid oxidation playing a central role in atherogenesis. The oxidation hypothesis of atherogenesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid and that are recognized by the innate immune system in animals and humans. These oxidized phospholipids are largely generated by potent oxidants produced by the lipoxygenase and myeloperoxidase pathways. The failure of antioxidant vitamins to influence clinical outcomes may have many explanations, including the inability of vitamin E to prevent the formation of these oxidized phospholipids and other lipid oxidation products of the myeloperoxidase pathway. Preliminary data suggest that the oxidation hypothesis of atherogenesis and the reverse cholesterol transport hypothesis of atherogenesis may have a common biological basis. The levels of specific oxidized lipids in plasma and lipoproteins, the levels of antibodies to these lipids, and the inflammatory/anti-inflammatory properties of HDL may be useful markers of susceptibility to atherogenesis. Apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides may both promote a reduction in oxidized lipids and enhance reverse cholesterol transport and therefore may have therapeutic potential.     

Oxidation of low-density lipoprotein in atherosclerosis from basic biochemistry to clinical studies

Although it has been known for long time that atherosclerosis is associated with lipid deposition, only recently it has been accepted that the plasmatic concentration of cholesterol, especially LDL cholesterol, is a risk factor for atherosclerosis. However, chemically modified LDL, but not native LDL, is able to induce the formation of foam cells, the hallmark of atherosclerosis. LDL oxidation is likely to be the most important form of LDL modification in humans. In biochemical terms, LDL oxidation is a free radical driven chain reaction where polyunsaturated fatty acids are converted to lipid peroxides, which easily decompose to many products, including biologically active aldehydes. The assay of LDL oxidation in biological fluids is problematic; direct assays detect a product of LDL oxidation whereas indirect assays give an indicator of LDL oxidation susceptibility. In general, epidemiological studies support the concept that the level of plasmatic lipophilic antioxidants, tocopherols and carotenoids, is low in populations at increased risk for atherosclerosis. However, clinical trials based on vitamin E as antioxidant showed inconclusive results, suggesting that supplementation with vitamin E is not generically recommended for atherosclerotic patients. These results, however, do not contradict that oxidation of lipoprotein is involved in atherosclerosis; rather, this negative outcome raises a number of considerations such as the need for a reliable marker of lipoprotein oxidation in plasma and a more complete information about the physiological triggers of lipoprotein oxidation.     

Use of a heptane-isopropanol system for extraction of primary lipid peroxidation products

Products of different lipids oxidation were studied for the peculiarities of their distribution in the extracting system heptane--isopropanol widely used in spectrophotometric determination of primary products of peroxide oxidation of lipids. It is shown that hydroperoxides of phospholipids and their reduction products almost completely transfer to the alcohol phase while hydroperoxides of triacetin and cholesterol esters are 70-85% extracted by heptane. Thus, it is necessary to analyze the alcohol phase when using the systems heptane-isopropanol to determine the content of primary products of lipid peroxide oxidation in those tissues, where phospholipids are the basic oxidation substrates.     

Oxysterols and mechanisms of apoptotic signaling: implications in the pathology of degenerative diseases

Oxysterols, or cholesterol oxidation products, are oxygenated derivatives of cholesterol which are formed endogenously during the biosynthesis of bile acids and steroid hormones. In addition, oxysterols may also be absorbed from the diet as they are found in many commonly consumed foods. Oxysterols have been shown to possess many potent and diverse biological activities, and the study of the effects of these oxidation products on the human body forms a wide field of research. The results of most research efforts support the conclusion that certain oxysterols, predominantly those found in oxidized low-density lipoprotein, exert pathological effects such as the induction of apoptotic cell death. Moreover, apoptosis induced by oxysterols has been strongly implicated in the pathogenesis of atherosclerosis as well as a variety of other diseases. The study of oxysterol-induced apoptosis is an emerging area, and the following review aims to provide a detailed account on the chronology of events involved. Current evidence of the involvement of the death receptor pathway and protein kinases is examined as well as important apoptosis regulators such as the mitochondria, B-cell lymphoma-2 proteins and caspases. The effect of oxysterols on gene expression, protein interactions and membrane properties are also discussed.     

The interactions of amphotericin B with various sterols in relation to its possible use in anticancer therapy.

Amphotericin B (AmB) is still the most common anti-fungal agent used to treat systemic fungal infections. It is known that this antibiotic acts by forming pores with the ergosterol contained in the membranes of fungi, but it also interacts with the cholesterol contained in the membranes of eukaryotic cells, hence its toxicity. AmB may also interact with the most common oxidation products of cholesterol found in vivo, together with interacting with biosynthetic precursors of cholesterol, namely, lanosterol and 7-dehydrocholesterol (7-DHC). The purpose of the present work was to study the interactions in solution between AmB and these various sterols, the techniques used being UV-Vis spectroscopy and differential scanning calorimetry. The results are globally interpreted in terms of the structural differences between the sterols. We show that AmB selectively interacts with 7-DHC which, according to a recent hypothesis proposed in the literature, has been identified in connexion with a therapeutic strategy against hepatocellular carcinomas. We find that the affinity of AmB towards 7-DHC is even greater than the affinity of the antibiotic towards ergosterol. We also find that AmB selectively interacts with the principal oxidation product of cholesterol, 7-ketocholesterol, a situation that has to be taken into account when AmB is administered.     

An experimental basis for carcinogenic effects of ultraviolet radiation

Several hypotheses to explain ultraviolet carcinogenesis have been advanced. One such theory contended that cholesterol was directly involved in actinic carcinogenesis. Although the hypothesis, in its original form, was generally abandoned by the scientific community, it has been revived and modified from time to time as structures and functions of steroid hormones become more clearly understood. In essence it suggests that carcinogenic substances, structurally related to steroid hormones might result from photochemical conversion of cholesterol. Although some compounds with such properties have been isolated under controlled chemical conditions, until recently the failure to find such compounds in biological systems had cast serious doubt upon the validity of this hypothesis. It has now been demonstrated that cholesterol-derived oxidation products are formed in human skin upon exposure to ultraviolet light. One of the products formed is known to possess carcinogenic properties when administered to experimental animals. Furthermore, it has been reported by other investigators that the control mechanism for cholesterol synthesis is absent in liver tumors. Whether this biochemical lesion plays a causal role in the etiology of this disease is unknown but altered cholesterol metabolism has also been implicated in actinically induced skin cancer. It has been demonstrated in this laboratory that skin sterol synthesis is inhibited by light. The principal site of action of light on sterol synthesis appears to be prior to the formation of acetyl coenzyme A in the biosynthetic pathway. Sterol-derived photoproducts produce similar effects as light upon sterol synthesis. These observations suggest more than just a coincidental role of light upon sterols and sterol metabolism in the etiology of skin cancer. Lunar Science Institute Contribution.     

Effect of mercury chloride on the level of lipids and products of their peroxidation in rat vital organs

The influence of mercury chloride on peroxidation processes of lipids and level of common lipids, phospholipids and spectrum of neutral lipids in liver, heart, lung and kidney of rats has been investigated. Administration of mercury chloride in a dose 0.7 mg/100 g of body weight to animals has invoked accumulation of lipids peroxide products in fractions of neutral lipids and phospholipids so it testifies the development of oxidative stress. Decrease of the most sensitive to oxidation fractions in the early stages of oxidative stress development and increase of free cholesterol and its ethers content in kidney and free cholesterol in the heart in more later terms as a result of mercury chloride administration have been revealed.     

On the mechanism of oxidation of cholesterol at C-7 in a lipoxygenase system.

Incubation of [7-2H2]cholesterol with soybean lipoxygenase and linoleic acid in the presence of oxygen gave a mixture of 5-cholestene-3 beta,7 alpha-diol, 5-cholestene-3 beta,7 beta-diol, 3 beta-hydroxy-5-cholesten-7-one,5 alpha,6 alpha-epoxycholestan-3 beta-ol, and 5 beta,6 beta-epoxycholestan-3 beta-ol. The conversion into the 7-oxygenated products was associated with a very high intermolecular isotope effect (KH/KD = 15-17), suggesting that the rate-limiting step in the overall conversion is likely to be the abstraction of hydrogen at C-7 in a radical reaction. Evidence that linoleic acid is to some extent directly involved was obtained with the use of [7-3H]cholesterol. Incubation of [7-3H]cholesterol resulted in a significant incorporation of 3H in the reisolated linoleic acid fraction. The isotope effect associated with conversion of [7 alpha-2H]cholesterol into 7-oxygenated products in the lipoxygenase system was 2-3, indicating that the extraction of hydrogen is nonstereospecific. Incubation of [7-2H2]cholesterol with 13-hydroperoxy-9,11-octadecadienoic acid gave the above 7-oxygenated products with relatively small isotope effects (KH/KD = 3-4). It is concluded that the most important mechanism for oxidation of cholesterol at C-7 in the lipoxygenase system involves participation of radicals and that a carbon-centered linoleic acid radical can extract hydrogen directly from cholesterol. Fatty acid hydroperoxides and their secondary products seem to be less important as initiators in connection with oxidation of cholesterol.     

Effect of membrane composition on lipid oxidation in liposomes

To study the effect of membrane composition on the oxidation of liposomes, different systems were prepared by adding one component at time to phosphatidylcholine (Epikuron 200). In particular, the effect of cholesterol and its ester, cholesterol stearate, on membrane structure and oxidation was studied. A first screening of the structure and net charge of the different preparation was made by means of z-potential and size measurements. Then the liposomes were oxidized by using a hydrophilic radical initiator, the (2,2-azobis(2-amidinopropane) hydrochloride, AAPH, which thermally decomposes to give a constant radical flux in water. The oxidation of liposomes, monitored by following the absorbance of the primary products of oxidation at 234 nm, was shown to be dependent on the composition of the liposomal bilayer and so on its biophysical properties. In addition, size and z-potential measurements gathered in the time course of the peroxidation reaction, revealed that the oxidation induced a modification of the superficial characteristics of the membrane bilayer so as to change its charge at the shear plane (z-potential). This behaviour was shared by all liposomal preparations independent of the composition. The change in sizes of the different liposomal preparation, instead, followed different trends, being more stable both in control samples and in oxidized ones when cholesterol was present. From the analysis of the results, it can be concluded that cholesterol affects the oxidation induced by hydrophilic radical initiator of model membranes by changing the biophysical properties of the phospholipid bilayer. The rigidity induced by cholesterol at temperatures above the T_m makes the membrane more resistant to radical attack from an external aqueous phase and this in turn delays the start of the reaction. The decrease of z-potential of the liposomal particles induced by the oxidation process can be an important clue to understand the mechanisms involved in the etiology of important diseases.