Cholesterol, Reactive Oxygen Species, and the Formation of Biologically Active Mediators

Abstract

Ch 2The abbreviations used are: Ch, cholesterol(s); ROS, reactive oxygen species; LDL, low density lipoprotein; DM, diabetes mellitus; ER, endoplasmic reticulum; SREBP, sterol regulatory element-binding protein. is a major lipid component in eukaryotic cells and can attain levels as high as 50% (on a molar basis) of all lipids present in the plasma membrane of certain cells (1Koumanov K.S. Tessier C. Momchilova A.B. Rainteau D. Wolf C. Quinn P.J. Arch. Biochem. Biophys. 2005; 434: 150-158Crossref PubMed Scopus (79) Google Scholar). The pathways responsible for Ch accumulation at this site are a consequence of gene regulation largely worked out by Brown and Goldstein (2Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). The very high concentration of this unique and singular lipid at the plasma membrane of cells places it in a position to encounter ROS that cross this permeability barrier. Ch can be oxidized in membranes even when polyunsaturated fatty acyl groups are not. Also, some unique products can serve as markers of certain ROS. The structure of Ch makes it susceptible to a variety of radical as well as nonradical oxidation reactions due to the 5,6-double bond and the concomitant vinylic methylene group at C-7 in the B ring. The isooctyl side chain at C-17 is a site for enzymatic oxidation largely by P450 cytochromes to form a series of biochemically active oxysterols, but this site of oxidation is typically not a target for ROS relevant to cellular biochemistry (3Smith, L. L. (1981) Cholesterol Autoxidation, Plenum Press, New YorkGoogle Scholar). Oxysterols are clearly formed in cells, but considerable evidence exists for a dietary origin of some oxysterols transported to cells by chylomicrons (4Vine D.F. Croft K.D. Beilin L.J. Mamo J.C. Lipids. 1997; 32: 887-893Crossref PubMed Scopus (62) Google Scholar). Various mechanisms, including heating of foods in air, can lead to formation of oxysterols that can be ingested. Several excellent reviews by Smith (3Smith, L. L. (1981) Cholesterol Autoxidation, Plenum Press, New YorkGoogle Scholar, 5Smith L.L. Chem. Phys. Lipids. 1987; 44: 87-125Crossref PubMed Scopus (283) Google Scholar) have covered this area, and although somewhat older literature, they are a wealth of information about Ch autoxidation. The free radical-based oxidation of Ch has been the focus of several biochemical studies (6Girotti A.W. J. Lipid Res. 1998; 39: 1529-1542Abstract Full Text Full Text PDF PubMed Google Scholar). The fairly complex mixture of oxysterols that result from free radical-mediated Ch oxidation confounds insight into the exact mechanism of formation of individual oxysterols. This is due in part to the random character of free radical oxidation as well as subsequent rearrangement of reactive intermediates. Four major products are typically observed, 7α,β-OOH-Ch, 7α,β-OH-Ch, 7-ox-Ch, and 5,6-epoxy-Ch (abbreviations and nomenclature are listed in supplemental Table 1), but many minor products, including A ring-oxidized species, are known (3Smith, L. L. (1981) Cholesterol Autoxidation, Plenum Press, New YorkGoogle Scholar, 5Smith L.L. Chem. Phys. Lipids. 1987; 44: 87-125Crossref PubMed Scopus (283) Google Scholar). The generation of highly reactive hydroxyl radicals (OH•) by various mechanisms, including H2O2 reduction by superoxide anion catalyzed by iron(II) (Fenton reaction), peroxynitrite (ONOO–) following the reaction of nitric oxide with superoxide anion, or ionizing radiation, can all eventually lead to abstraction of a C-7 allylic hydrogen atom, forming a carbon-centered radical at this site. The focus of these free radical oxidations of Ch is at C-7 because the carbon–hydrogen bond is sufficiently weak at this position (bond dissociation energy of 88 kcal/mol (7Gardner H.W. Free Radic. Biol. Med. 1989; 7: 65-86Crossref PubMed Scopus (586) Google Scholar)) that longer lived lipid peroxy radical or alkoxyl radicals derived from polyunsaturated fatty acyl lipid peroxyl radicals (LOO•) can readily abstract this hydrogen atom. The C-7-centered radical intermediate of Ch has a sufficiently long half-life to encounter a ground state molecular oxygen molecule, forming a hydroperoxy radical. This means that the propagation of free radical chemistry through the lipid bilayer, which might be initiated at some point by hydroxyl radical, can lead to the observed peroxidation of Ch. This hydroperoxy radical intermediate can abstract certain hydrogen atoms and thus propagate the free radical reaction, forming (after hydrogen abstraction) chemically stable 7α- and 7β-OOH-Ch (Fig. 1). Interestingly, recent evidence has shown that Ch hydroperoxides move much more efficiently between various cellular compartments compared with Ch itself (8Vila A. Levchenko V.V. Korytowski W. Girotti A.W. Biochemistry. 2004; 43: 12592-12605Crossref PubMed Scopus (42) Google Scholar) and thus become available for many sites within the cell for further reactions, including reductions by glutathione-dependent selenoperoxidases (6Girotti A.W. J. Lipid Res. 1998; 39: 1529-1542Abstract Full Text Full Text PDF PubMed Google Scholar) or by nonenzymatic reactions with chelated transition metals (7Gardner H.W. Free Radic. Biol. Med. 1989; 7: 65-86Crossref PubMed Scopus (586) Google Scholar). Reduction of 7α,β-OOH-Ch by iron(II) can lead to a 7α,β-alkoxyl-Ch radical that can undergo several different pathways of reaction. For example, hydrogen abstraction from another molecule results in continued free radical propagation and formation of stable 7α,β-OH-Ch. The formation of the 7-oxo-Ch product is thought to be due in part to the reaction of this 7α,β-alkoxyl radical with another lipid peroxy radical, which abstracts the remaining proton at C-7 and forms the 7-oxo product in a radical propagation termination reaction (9Chang Y.H. Abdalla D.S.P. Sevanian A. Free Radic. Biol. Med. 1997; 23: 202-214Crossref PubMed Scopus (92) Google Scholar). Another mechanism for the formation of the typically abundant 7-oxo-Ch can derive from interaction of two hydroperoxy radicals in a "Russell mechanism" (10Howard J.A. Ingold K.U. J. Am. Chem. Soc. 1968; 90: 1056-1058Crossref Scopus (243) Google Scholar) and a cyclic tetroxide intermediate (Fig. 1). The products of this reaction would be 7-oxo-Ch, a hydroxy lipid, and singlet oxygen (1O2) to conserve electronic spin states in this mechanism (10Howard J.A. Ingold K.U. J. Am. Chem. Soc. 1968; 90: 1056-1058Crossref Scopus (243) Google Scholar). 7-Oxo-Ch can also result from enzymatic oxidation of 7-OH-Ch (11Niki E. Yoshida Y. Saito Y. Noguchi N. Biochem. Biophys. Res. Commun. 2005; 338: 668-676Crossref PubMed Scopus (643) Google Scholar). These multiple pathways may help to explain the abundance of this product. Radical hydrogen atom abstraction at a bisallylic methylene group found in polyunsaturated fatty acyl moieties of phospholipids would be expected to be energetically more favorable relative to the C-7 methylene group in Ch. Indeed, in plasma, the formation of oxidized polyunsaturated fatty acyl products was observed to be 30-fold higher than that of oxidized Ch (12Yoshida Y. Niki E. Free Radic. Res. 2004; 38: 787-794Crossref PubMed Scopus (53) Google Scholar), yet in a lipid bilayer, the structured environment (entropy) can alter the propagation path of free radical events, likely as a result of the location of radicals with respect to ordered Ch. Studies have been carried out in cells in which it was observed that phospholipids containing polyunsaturated fatty acids were less susceptible to oxidation (13Saito Y. Yoshida Y. Niki E. FEBS Lett. 2007; 581: 4349-4354Crossref PubMed Scopus (32) Google Scholar); however, plasmalogen phospholipids were found to inhibit Ch peroxidation (14Maeba R. Ueta N. J. Lipid Res. 2003; 44: 164-171Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). These studies have revealed extensive oxidation of Ch prior to oxidation at the bisallylic methylene groups of the phospholipids. The propagation of radical events through a lipid bilayer is likely the mechanism by which Ch eventually becomes oxidized at C-7 due to the high abundance of this single lipid. Other products of Ch radical oxidation are the epimeric 5,6-epoxides (Fig. 1). Both 5α,6α- and 5β,6β-epoxy-Ch are observed as products. Interestingly, these products require an intermediate formation of hydroperoxy lipids and are not the primary products of oxidation at C-7 of Ch (3Smith, L. L. (1981) Cholesterol Autoxidation, Plenum Press, New YorkGoogle Scholar). The formation of these epoxy-Ch metabolites can be a result of a radical-based mechanism of attack of LOO• at the Δ5,6-double bond, forming an initial radical adduct at C-5 or C-6 (Fig. 1) followed by loss of an alkoxyl lipid radical (15Watabe T. Tsubaki A. Isobe M. Ozawa N. Hiratsuka A. Biochim. Biophys. Acta. 1984; 795: 60-66Crossref PubMed Scopus (11) Google Scholar). A nonradical-based mechanism for the formation of these epoxy-Ch has been proposed based upon the known epoxidation of olefins by hydroperoxides (3Smith, L. L. (1981) Cholesterol Autoxidation, Plenum Press, New YorkGoogle Scholar, 4Vine D.F. Croft K.D. Beilin L.J. Mamo J.C. Lipids. 1997; 32: 887-893Crossref PubMed Scopus (62) Google Scholar). These 5,6-epoxides are observed as the most abundant nonenzymatically formed oxysterols in the macrophage. 3D. Russell, personal communication. This nonradical mechanism has been suggested to account for the occurrence of epoxy-Ch derived from Ch esters in oxidized LDL. In this case, the initial peroxidation site is at the fatty acyl moiety such as the linoleoyl acyl group and initial formation of a linoleoyl hydroperoxide. This hydroperoxide at C-9 or C-13 of the fatty acyl chain could rearrange, intramolecularly, to the corresponding epoxy-Ch with the hydroperoxy group reduced to a hydroxy moiety (16Spiteller G. Free Radic. Biol. Med. 2006; 41: 362-387Crossref PubMed Scopus (155) Google Scholar). Several nonradical pathways of Ch peroxidation can occur in cellular membranes that are not dependent upon oxygen- or carbon-centered radicals that do not result in free radical propagation reactions through phospholipids, at least initially. One widely studied reaction has been that with singlet oxygen (1ΔgO2, abbreviated 1O2 is on the order of a few microseconds, this reactive form of oxygen can travel only a fraction of the cell diameter of a typical eukaryotic cell, yet singlet oxygen readily produces one major and two minor products by an "ene" reaction mechanism with Ch (Fig. 2). The reaction of singlet oxygen to form 5α-OOH-Ch has been found to have the highest rate constant relative to formation of 6α,β-OOH-Ch. Furthermore, the 5α-OOH-Ch product is metabolized at a lower rate, which helps explain the accumulation of this oxidized metabolite of Ch when singlet oxygen is exposed to cells (18Korytowski W. Girotti A.W. Photochem. Photobiol. 1999; 70: 484-489Crossref PubMed Scopus (49) Google Scholar). The initially formed 5α-OOH-Ch is also known to rearrange to 7α,β-OOH-Ch upon sample work-up and possibly in situ in the membrane (6Girotti A.W. J. Lipid Res. 1998; 39: 1529-1542Abstract Full Text Full Text PDF PubMed Google Scholar, 19Beckwith A.L.J. Davies A.G. Davison I.G.E. Maccoll A. Mruzek M.H. J. Chem. Soc. Perkin Trans. I. 1989; 2: 815-824Crossref Google Scholar). These observations have led to the suggestion that measurement of 5α-OOH-Ch could be a marker for singlet oxygen production within cells (17Girotti A.W. Korytowski W. Methods Enzymol. 2000; 319: 85-100Crossref PubMed Google Scholar). The reaction of Ch with ozone has been known for some time to be both rapid and complex (3Smith, L. L. (1981) Cholesterol Autoxidation, Plenum Press, New YorkGoogle Scholar). The lung is particularly susceptible to ozone due to the presence of ozone in the troposphere and therefore present in air inspired into the lung. Recent studies have revealed the formation of two major products when low concentrations (100–500 ppb) of ozone (such levels can be present in the air of large cities, to which humans are exposed on a constant basis) were exposed to lung surfactant (20Pulfer M.K. Murphy R.C. J. Biol. Chem. 2004; 279: 26331-26338Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The initial reaction of ozone is the addition across the Δ5-double bond of Ch, which yields a 1,2,3-trioxolane that undergoes rapid and spontaneous decomposition to yield several additional reactive entities, including carbonyl oxides, carbon-centered radicals, and hydroperoxides, which ultimately lead to the cleavage of the C-5–C-6 double bond (Fig. 2). Some of the more stable ozonide products have been recently structurally characterized (21Pulfer M.K. Harrison K. Murphy R.C. J. Am. Soc. Mass Spectrom. 2004; 15: 194-202Crossref PubMed Scopus (17) Google Scholar). Peroxidation of polyunsaturated fatty acyl groups is also initiated by reaction of ozone, leading to the formation of abundant phospholipid hydroperoxides. The second major product of Ch when ozone was exposed to surfactant was identified as 5,6β-epoxy-Ch, which could be formed by rearrangement of the 1,2,3-trioxolane intermediate (Fig. 2) or by a phospholipid or Ch hydroperoxide mechanism as described above (Fig. 1). Recently, a set of Ch ozonolysis products was described as being formed in atherosclerotic plaques as a result of in situ formation of ozone (22Wentworth P.J. Nieva J. Takeuchi C. Galve R. Wentworth A.D. Dilley R.B. DeLaria G.A. Saven A. Babior B.M. Janda K.D. Eschenmoser A. Lerner R.A. Science. 2003; 302: 1053-1056Crossref PubMed Scopus (241) Google Scholar). Although there is some controversy about the identification of the products and mechanism of formation of ozone by this model (23Smith L.L. Free Radic. Biol. Med. 2004; 37: 318-324Crossref PubMed Scopus (66) Google Scholar), nonetheless, these products do possess potent activities on cells. These metabolites, atheronals A and B (Fig. 2), would result from the expected cleavage of the B ring and aldol condensation (22Wentworth P.J. Nieva J. Takeuchi C. Galve R. Wentworth A.D. Dilley R.B. DeLaria G.A. Saven A. Babior B.M. Janda K.D. Eschenmoser A. Lerner R.A. Science. 2003; 302: 1053-1056Crossref PubMed Scopus (241) Google Scholar, 24Wang K. Bermudez E. Pryor W.A. Steroids. 1993; 58: 225-229Crossref PubMed Scopus (42) Google Scholar). The abundant production of HOCl by neutrophils and HOBr by eosinophils leads to the potential exposure of these very ROS to Ch during phagocytosis. Hypochlorous acid is known to react with olefins to form chlorohydrins by simple electrophilic addition across the double bond. Such oxidation products have been found following reaction with HOCl (25van den Berg J.J. Winterbourn C.C. Kuypers F.A. J. Lipid Res. 1993; 34: 2005-2012Abstract Full Text PDF PubMed Google Scholar). Unexpectedly, there was evidence for the addition of two chlorine atoms across the 5,6-double bond of Ch from Cl2, yielding a very nonpolar 5,6-dichlorocholestane metabolite. When fatty streaks from atherosclerotic plaques were examined, this metabolite was readily detected (26Hazen S.L. Hsu F.F. Duffin K. Heinecke J.W. J. Biol. Chem. 1996; 271: 23080-23088Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). The recognition that Ch and Ch esters can be oxidized by nonenzymatic means has driven two very separate types of studies: (i) to assess the involvement of ROS in a disease process and to identify marker products and (ii) to assess the role of oxysterols in a disease process by way of mediating biological responses. The first type of study demands specific and sensitive analytical capability, which has substantially improved in the past decade with the development of electrospray ionization mass spectrometry and liquid chromatography/tandem mass spectrometry. It was not possible to analyze species such as the hydroperoxides of Ch directly by gas chromatography/mass spectrometry. Now even reactive hydroxy, hydroperoxy, and intact ozonides can be directly analyzed using electrospray ionization (21Pulfer M.K. Harrison K. Murphy R.C. J. Am. Soc. Mass Spectrom. 2004; 15: 194-202Crossref PubMed Scopus (17) Google Scholar). This capability has changed both types of oxysterol studies because it is now possible to detect those oxysterol intermediates and to focus attention on additional oxysterol entities that could mediate biological activities. Recent examples include measurement of the specific oxysterols that are formed in both type 1 and type 2 DM in kidney, heart, and liver (27Yoshioka N. Adachi J. Ueno Y. Yoshida K. Free Radic. Res. 2005; 39: 299-304Crossref PubMed Scopus (29) Google Scholar). It was found that levels of 7α- and 7β-OOH-Ch as well as 7α- and 7β-OH-Ch were increased in all tissues analyzed, consistent with initiation of free radical lipid peroxidation. The plasma of patients with DM has been studied, and the oxysterols 7α- and 7β-OH-Ch, and 5α,6α- and 5β,6β-epoxy-Ch were measured. These oxysterols increased in the DM plasma, and these oxysterols have been suggested to be useful as biomarkers for oxidative damage in patients with DM (28Ferderbar S. Pereira E.C. Apolinario E. Bertolami M.C. Faludi A. Monte O. Calliari L.E. Sales J.E. Gagliardi A.R. Xavier H.T. Abdalla D.S. Diabetes Metab. Res. Rev. 2007; 23: 35-42Crossref PubMed Scopus (57) Google Scholar). Studies probing the biological action of oxysterols had been an active area of investigation, and many aspects have been reviewed extensively (29Smith L.L. Johnson B.H. Free Radic. Biol. Med. 1989; 7: 285-332Crossref PubMed Scopus (334) Google Scholar, 30Bjorkhem I. Diczfalusy U. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 734-742Crossref PubMed Scopus (257) Google Scholar, 31Massey J.B. Curr. Opin. Lipidol. 2006; 17: 296-301Crossref PubMed Scopus (71) Google Scholar). Recent studies have focused considerably on the role oxysterols play as mediators in atherosclerosis, cytotoxicity, and regulation of Ch biosynthesis. Many studies have now established that various oxysterols can be found in fatty streaks, aortic plaques, and even aortic tissue of human atherosclerotic plaques (32Brown A.J. Jessup W. Atherosclerosis. 1999; 142: 1-28Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar). The post-mortem analysis of aortic tissue from rabbits fed a high Ch diet revealed the presence of several oxysterols at increased levels in atherosclerotic areas (33Hodis H.N. Crawford D.W. Sevanian A. Atherosclerosis. 1991; 89: 117-126Abstract Full Text PDF PubMed Scopus (148) Google Scholar). In human studies, 7α- and 7β-OH-Ch, 5,6-epoxy-Ch, and 7-oxo-Ch were found at substantially higher levels in fatty streaks and in advanced lesions compared with normal tissues (34Garcia-Cruset S. Carpenter K.L. Guardiola F. Stein B.K. Mitchinson M.J. Free Radic. Res. 2001; 35: 31-41Crossref PubMed Scopus (121) Google Scholar). These observations led to detailed studies of the biological activity of certain oxysterols; however, their involvement in atherosclerosis remains unclear. Early stage atherosclerosis involves conversion of the macrophage to foam cells due in part to the uptake of oxidized LDL. Detailed studies of the lipids present in oxidized LDL have led to identification of a host of different oxidized phospholipids as well as oxysterols. Therefore, probing the activity of specific oxysterols added to endothelial cells has been a logical experiment considering that ROS-derived oxysterols are cytotoxic and may induce apoptosis. However, these pharmacological studies in many high concentrations of a single which is to occur in In when of oxysterols at relevant concentrations were added to cells, it was to biological activity G. F. E. G. Med. 2004; PubMed Scopus Google Scholar). Recently, two specific oxysterols derived from the reaction of ozone with Ch have been proposed to play a role in the of atherosclerosis (22Wentworth P.J. Nieva J. Takeuchi C. Galve R. Wentworth A.D. Dilley R.B. DeLaria G.A. Saven A. Babior B.M. Janda K.D. Eschenmoser A. Lerner R.A. Science. 2003; 302: 1053-1056Crossref PubMed Scopus (241) Google Scholar). studies of these products, atheronals A and with human endothelial cells, and human revealed that both and of the cells the oxysterols C. Galve R. Nieva J. Wentworth A.D. Lerner R.A. Wentworth Biochemistry. 2006; PubMed Scopus Google Scholar). A can also increased of the molecule and B of into C. Galve R. Nieva J. Wentworth A.D. Lerner R.A. Wentworth Biochemistry. 2006; PubMed Scopus Google Scholar). Studies of the activities of various oxysterols have led to the of biological activities relevant to atherosclerosis Med. 2001; PubMed Scopus Google Scholar). various oxysterols can lead to macrophage cell in the of atherosclerosis I. 2005; PubMed Scopus Google Scholar). 7-oxo-Ch, 5,6-epoxy-Ch, and have been shown to although at high concentrations J.A. Thromb. Res. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Although the molecular by which these oxysterols these activities are such studies do present as to the potential role that oxysterols, derived from nonenzymatic reactions of Ch with ROS in may play in a disease as complex as One of the of nonenzymatically derived oxysterols is the on cell studies have revealed that 7β-OH-Ch, 7-oxo-Ch, and 5β,6β-epoxy-Ch are cytotoxic at low concentrations most likely by cellular S. C. T. A. A. G. L. D. G. Biol. 2005; PubMed Scopus Google Scholar). The possible mechanism for of in cells cells may be to the of membranes within the cells such as the J.B. Curr. Opin. Lipidol. 2006; 17: 296-301Crossref PubMed Scopus (71) Google Scholar). various oxysterols, including 7β-OH-Ch, 7-oxo-Ch, and 5,6-epoxy-Ch, are found in oxidized LDL, pharmacological studies of these have been carried in the observations that these could the cytotoxic of oxidized LDL G. S. S. S. D. FEBS Lett. 1997; PubMed Scopus Google Scholar). In addition to the Ch products initially formed by ROS, subsequent enzymatic can For example, 5β,6β-epoxy-Ch is a for to yield the corresponding J.W. C. Lipid Res. 2005; 44: PubMed Scopus Google Scholar). This metabolite has activity K. Biochim. Biophys. Acta. 2006; PubMed Scopus Google Scholar) and can be also metabolized to a unique metabolite that was found in cells as M.K. C. E. Murphy R.C. J. 2005; PubMed Scopus Google Scholar). This derived oxysterol was found to have considerable biological activity and for the of the activity of in cells in of both and Ch biosynthesis. Detailed studies of the mechanism of cellular by oxysterols, 7-oxo-Ch, have been reviewed M.S. Curr. Opin. Lipidol. 2001; PubMed Scopus Google Scholar). In it is thought that these oxysterols induce cellular through the of from the and of or through the It has been known for some time that oxysterols are of Ch (3Smith, L. L. (1981) Cholesterol Autoxidation, Plenum Press, New YorkGoogle Scholar). Although many of the products of nonenzymatic oxidation of Ch can inhibit (29Smith L.L. Johnson B.H. Free Radic. Biol. Med. 1989; 7: 285-332Crossref PubMed Scopus (334) Google Scholar), only recently has a more detailed the of this It is thought that the of Ch by itself and oxysterols by way of formation of a from For the active to be and travel into the the to another and this complex the and be transported to the This process involves to which the complex into that from the membrane and through become with the A. Brown M.S. Goldstein J.L. 2004; 15: Full Text Full Text PDF PubMed Scopus Google Scholar). Ch this by to to two and of the component of the complex with the J. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). has to the the complex move to the Recent evidence has suggested that some oxysterols, the formed to the than the J. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). It is not however, a role is by the nonenzymatically formed oxysterols because they were intermediate in in to or of these detailed studies was the that Ch and oxysterols were not the of the but their through with specific A. Y. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar, L. A. Brown M.S. Goldstein J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A large of oxysterols can be by the various ROS, but few have been in to or the It is possible that some oxidized Ch products from the nonenzymatic could have different in when considering the more reactive intermediate such as the hydroperoxy and subsequent metabolites by of oxysterols within the The nonenzymatic oxidation of Ch leads to a host of oxysterols, the of which are largely dependent upon the ROS that the of Ch. When ROS mediate free radical oxidation of Ch, a very and complex mixture of oxysterols results from both the initial species of Ch and their subsequent metabolites, which can be formed within the Other ROS, including ozone, are in of the of the initial Ch intermediates as well as activities of subsequent These may their activity through a of the membrane on the of both lipids and within It is also possible that these to regulatory in Ch or lipid biochemical These and may be through a interaction with or A detailed of the role that various oxysterols play in disease is not at largely because of the of the mixture of oxysterols present in the potential interaction of various and the observed of the cell to the oxysterols. in being to the of the Ch by ROS have been which further of the and concentrations of different products and insight into the role that these play in