Review
Pathways of cholesterol oxidation via non-enzymatic mechanisms

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Abstract

Cholesterol has many functions, including those that affect biophysical properties of membranes, and is a precursor to hormone synthesis. These actions are governed by enzymatic pathways that modify the sterol nucleus or the isooctyl tail. The addition of oxygen to the cholesterol backbone produces its derivatives known as oxysterols. In addition to having an enzymatic origin, oxysterols can be formed in the absence of enzymatic catalysis in a pathway usually termed “autoxidation,” which has been known for almost a century and observed under various experimental conditions. Autoxidation of cholesterol can occur through reactions initiated by free radical species, such as those arising from the superoxide/hydrogen peroxide/hydroxyl radical system and by non-radical highly reactive oxygen species such as singlet oxygen, HOCl, and ozone. The susceptibility of cholesterol to non-enzymatic oxidation has raised considerable interest in the function of oxysterols as biological effectors and potential biomarkers for the non-invasive study of oxidative stress in vivo.

Highlights

• Cholesterol has many functions. • Addition of oxygen into the cholesterol backbone produces derivatives known as oxysterols. • Oxysterols are produced by enzymatic mechanisms or by autoxidation. • Autoxidation of cholesterol occurs via free radical or non-radical pathways.

Introduction

Cholesterol (Chol) is located in all membrane compartments at levels as high as 50-mole percent, which renders it the most prominent lipid in eukaryotic cells (Bloch, 1983). Cholesterol, which is synthesized de novo and obtained from the diet, largely affects the biophysical properties of cellular membranes and functions in a variety of synthetic pathways, including those of bile acids, hormones, and, through its precursor 7-dehydrocholesterol (7-DH-Chol), vitamin D synthesis. Most of these actions are associated with cholesterol oxidation occurring either via enzymatic or non-enzymatic mechanisms. Certain oxysterols are produced exclusively by autoxidation of cholesterol, whereas others are solely produced through enzymatic mechanisms. However, most oxysterols found in biological fluids can be produced by both autoxidation and enzymatic mechanisms. The addition of oxygen to the cholesterol backbone creates a class of derivatives known as oxysterols (Fig. 1). The addition of oxygen may occur via non-enzymatic or enzymatic mechanisms.

The term autoxidation is seldom used to refer to non-enzymatic cholesterol oxidation. This term is potentially misleading because cholesterol appears to oxidize by itself without the aid of factors other than oxygen in the ground state. This type of autoxidation involves oxygen present in the air, but the initiation of the autoxidation process requires one or more additional factors, including trace amounts of metals and reactive oxygen species. In in vitro systems, the presence of transition metals can be controlled for; however, trace amounts of peroxides or other forms of reactive oxygen species may still be present at undetectable levels and could be responsible for the initiation process. In biological systems, given the complexity of the milieu, the initiator could easily be a transition metal or a reactive oxygen species, like ozone and singlet oxygen, which are present in the atmosphere and at sites of inflammation, respectively. Therefore, the term “non-enzymatic oxidation” might be more appropriate than the potentially misleading term, “autoxidation.” However, autoxidation is still a valid and simple term for describing all process outside the enzymatic pathways of cholesterol transformation. Thus, autoxidation and non-enzymatic oxidation will be used interchangeably throughout this text. Cholesterol autoxidation has been known for almost a century and has been observed under a variety of experimental conditions. Noteworthy, commercially obtained cholesterol can autoxidize in air while still in the manufacturer's shipping container. In advanced oxidation, the compound develops a characteristic odor, which is caused by fragments that are derived from alyphatic side-chain breakage, as well as a yellowish coloration. Cholesterol can be oxidized through reactions that are initiated by free radical species, of which the most studied are those initiated by the superoxide/hydrogen peroxide/hydroxyl radical system, from nitric oxide, and by non-radical reactive oxygen species such as singlet oxygen (1ΔgO2), HOCl, and ozone (O3).

Regarding enzymatic oxysterols, there are numerous cholesterol metabolizing enzymes, many of which belong to the cytochrome P450 (CYP) family (Pikuleva, 2008). Cholesterol 7α-hydroxylase (CYP7A1) is a hepatic enzyme that converts cholesterol into 7α-hydroxycholesterol (7α-OH-Chol), one of the first intermediates in the biosynthesis of bile acids (Björkhem and Eggertsen, 2001). A portion of the 7α-OH-Chol formed in the liver leaks out into the circulatory system, and the concentration of 7α-OH-Chol in blood plasma correlates with the activity of CYP7A1 (Björkhem et al., 1987). 7α-OH-Chol is converted next to 7alpha-hydroxy-4-cholesten-3-one by a microsomal 3β-hydroxy-Δ5-C27-steroid oxidoreductase (C27 3β-HSD), which can be used as a marker of bile acid synthesis (Heverin et al., 2007). The drug-metabolizing enzyme CYP3A4 converts cholesterol into 4β-hydroxycholesterol (Bodin et al., 2001), an oxysterol that can be used as an endogenous marker of CYP3A4 and CYP3A5 activity (Diczfalusy et al., 2008). The brain-specific cholesterol 24-hydroxylase (CYP46A1) converts cholesterol into 24S-hydroxycholesterol (24-OH-Chol), a form of cholesterol that can be transported across the blood–brain barrier and which is important for maintaining cholesterol homeostasis in the brain (Lutjohann et al., 1996, Ohyama et al., 2006). The mitochondrial enzyme sterol 27-hydroxylase (CYP27A1), which is important for the side-chain oxidation of cholesterol 7α-hydroxylated intermediates during bile acid biosynthesis (Javitt, 2002), is broadly expressed in tissues and converts cholesterol to 27-hydroxycholesterol (27-OH-Chol). CYP27A1 produces 27-hydroxy-derivatives of cholesterol precursors, including lanosterol, zymosterol, desmosterol, and 7-dehydrocholesterol (Javitt, 2004).

25-Hydroxycholesterol (25-OH-Chol) is formed as a by-product of CYP27A1 (Lund et al., 1993) and by a specific hydroxylase, cholesterol 25-hydroxylase (Chol-25H), which is not a cytochrome P450 enzyme. This enzyme belongs to a group of enzymes that utilize oxygen and diiron as cofactors to catalyze hydroxylation reactions (Lund et al., 1998). The oxysterol 24(S), 25-epoxycholesterol (24,25-epoxy-Chol) is produced during cholesterol synthesis by the enzyme oxidosqualene cyclase (Wong et al., 2007). The susceptibility of cholesterol to non-enzymatic oxidation has generated considerable interest in oxysterols as potential markers for the non-invasive study of oxidative stress in vivo. Additional interest in oxysterols stems from the biological activity of many oxysterols, which can be useful for elucidating the pathophysiological pathways of human diseases and for the development of pharmacological tools.

This paper focuses on cholesterol autoxidation in an attempt to provide a summary of the oxysterols arising from non-enzymatic sources. Oxysterol research covers multiple aspects, including separation, identification, formation, metabolism, and levels of oxysterols in biological fluids. A fairly comprehensive review of the various aspects of oxysterol research has been published by Schroepfer (2000). I have not included plant oxysterols, which are vehicled in the organism by diet, an emerging area of intense investigation (for a review see Otaegui-Arrazola et al., 2010).

Section snippets

Non-enzymatic cholesterol oxidation: historical perspectives

More than a century ago, cholesterol was shown to autoxidize upon exposure to air (Schültz and Winterstein, 1904) and to produce oxidized products upon heating in the presence of benzoyl peroxide (Lifschütz, 1907). These products, which are blue/green in color, enabled the development of the first colorimetric test for oxidized cholesterol. Subsequently, extensive studies have established the crucial role of oxygen in the mechanism of cholesterol autoxidation (Blix and Löwenhielm, 1928). In

Plausibility of the biological activity of oxysterols

Oxysterols exert a multitude of biological effects of potential pathophysiological relevance, which are mediated by their biophysical effects on membranes and/or stereospecific interactions with proteins. Oxysterols can be viewed as the end products of free radical reactions and as a part of the cellular machinery that governs the integrity and function of the cell by acting at the level of signaling and translational- and post-translational-gene expression.

Oxysterols have a peculiar

Oxysterols as markers of oxidative stress

Oxidative stress can be defined as the steady state condition where the free radical/highly reactive species flux is balanced by antioxidant defenses. Oxidative stress occurs in healthy organisms but can be upregulated when the flux of free radicals/highly reactive species increases and/or antioxidants are reduced.

Given their short life span, free radicals and highly reactive species cannot be measured directly, and it is necessary to analyze the products that arise from reactions caused by

Perspectives

Although autoxidation of cholesterol has been known for over a century, the relevance of oxysterols in biology and medicine has been appreciated by and confined to a research niche. In the field of lipid peroxidation, the role of cholesterol has always been overtaken by that of PUFAs, most likely due to the presumed higher oxidizability of PUFAs due to the presence of multiple labile hydrogens. In the last few decades, the sharp increase in the number of LDL oxidation studies in the context of

Acknowledgements

The author is grateful for discussions with all members of the European Network on Oxysterols Research (ENOR) (www.oxysterolsnet.org) and is particularly indebted to participants of the LipidomicNet-ENOR joint Workshop in Munich in November 2010 for their discussions and encouragement.

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