I. INTRODUCTION

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Oxysterols represent a class of potent regulatory molecules with remarkably diverse, important biological actions. Early research on oxysterols concerned their generation from cholesterol (Chol) by autoxidation. Important studies by Bergström and Wintersteiner (64-66) and by others (681), reviewed by Bergström and Samuelsson (63), were extensively expanded by Smith and colleagues (1000, 1001). The ease of autoxidation of Chol noted in these important papers appears to have been ignored in the design and/or interpretation of many recent studies. Other investigations concentrated on selected oxysterols as a part of studies attempting to elucidate pathways and individual reactions involved in the formation of bile acids. These studies, spanning many decades, continue today. A major milestone was the demonstration by Kandutsch and Chen (466, 467) that certain oxygenated derivatives of Chol, but not highly purified Chol itself, caused an inhibition of sterol biosynthesis and a lowering of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in cultured mammalian cells. These findings stimulated a tremendous amount of research on oxysterols, including their actions on a wide variety of other processes and the chemical preparation of a large number of natural and synthetic oxygenated sterols, leading to the demonstration that some 15-oxygenated sterols were not only extraordinarily potent in suppressing sterol synthesis in cultured cells but also showed significant hypocholesterolemic action upon administration to rodents and nonhuman primates. An increasing amount of evidence indicates that oxysterols represent major natural regulators of sterol synthesis and of HMG-CoA reductase. Seminal studies by Brown and Goldstein (139, 140, 142) defined the metabolic defect in familial hypercholesterolemia (FH) and identified the receptor for low-density lipoprotein (LDL). These discoveries resulted in an explosion of research on LDL, including its roles in the control of sterol biosynthesis and a large variety of other important cellular processes, and studies on the levels of oxysterols in LDL. A considerable amount of research has been focused on the possible involvement of oxysterols, as components of oxidatively modified LDL, in the pathogenesis of atherosclerosis. Recent research has shown that oxysterols can induce programmed cell death in a variety of cell types and can also affect the development of calcification in vascular cells. Brown and Goldstein and their associates have recently discovered extraordinarily complex mechanisms involved in the processing of transcription factors that affect genes for critical enzymes involved in Chol biosynthesis. In addition, the same transcription factors have also been shown to be involved in biosynthesis of other important lipids. Oxysterols have also been reported to affect other transcription factors including several nuclear orphan receptors, and specific oxysterols may represent their natural ligands. Despite continuing advances in studies of the chemistry of oxysterols, most investigators have relied on commercial materials that are unfortunately very limited with regard to structural types, available quantities, and reasonable costs. This situation has resulted in the acquisition of a large amount of information on the effects of one oxysterol, 25-OH-Chol, on a wide variety of parameters in cultured mammalian cells. Unfortunately, other oxysterols may be of considerably more physiological importance. Moreover, the results of studies with 25-OH-Chol (or the combination of 25-OH-Chol and Chol) have been frequently generalized to other oxysterols without experimentation. The limited availability of oxysterols is also a major factor responsible for the very restricted number of studies of their in vivo effects in animals.

Knowledge of the metabolism of individual oxysterols is very important in the interpretation of the effects of their addition to a given cell type in tissue culture studies or of their administration to animals. For example, administration of one oxysterol, 3-hydroxy-5-cholest-8(14)-en-15-one, to animals is associated with very rapid and substantial conversion to a complex array of polar metabolites of varying potencies in their biological actions. Moreover, construction of analogs in which the major in vivo metabolism of the 15-ketosterol is blocked gave compounds with improved potency with regard to hypocholesterolemic action and other effects. To understand the potential physiological or pathophysiological importance of oxysterols in biology and medicine, many investigations have focused on the determination of the levels of oxysterols in blood plasma, in plasma lipoproteins, various tissues, and food preparations. High levels of certain oxysterols have been reported in cataracts, in membranes of red blood cells from patients with sickle cell disease, and in plasma from patients with liver disorders. Major advances have been made in the technologies for the separation, identification, and quantitation of oxysterols. Unfortunately, a good deal of the information currently available on the levels (and even identity) of oxysterols is of dubious validity due to lack of appreciation and/or lack of attention to critical aspects in the application of existing methodologies. Oxygenated sterols have significant potential for applications in medicine because of their effects on Chol metabolism, cell growth (both of normal and transformed cells), and other processes.

I have attempted to provide a critical, fairly comprehensive review of important, very rapidly expanding, multiple areas of investigation on oxysterols. My coverage concentrates on mammalian systems and, with a few exceptions, does not include oxysterols in plants and lower forms. The review is offered in a spirit of attempting to assist young, and not so young, investigators seeking to initiate or expand research on oxysterols. Over 25 years of participation in investigations on oxysterols provides some measure of personal experience on many, but not all, of the topics covered. Restriction to a review of our own studies would have been a relatively simple task. Past personal experience with reviews of broad fields of research (313, 908, 909, 910) provided the recognition of the effort and masochism required. Nonetheless, the present review furnished a special challenge that I hope will be useful to others.

This review begins with a coverage of methods for the separation and identification of oxysterols, a topic of considerable practical importance for research with these compounds. It is hoped that this coverage facilitates the selection and critical employment of the various methodologies. Sections on the formation, occurrence, and metabolism of the oxysterols are very important in understanding the actions and physiological significance of the various oxysterols. Critical coverage of some of these matters (with considerations of details of methodology) is provided not merely to correct errors but also to avoid perpetuation of the same in future research. Sections relating to the effects or activities of oxysterols include provision of their precise concentrations so as to allow the reader to appreciate the potencies of some of the oxysterols and to judge the potential physiological relevance of the reported observations.

As with many areas of research, some specialized nomenclature is involved. Presented in Figure 1 is the chemical structure and numbering system for cholestane. To conserve space, cholesterol is abbreviated as Chol, and hydroxy derivatives are presented as OH-Chol, e.g. 25-OH-Chol for 25-hydroxycholesterol. 5,6-Epoxy-Chol, 5,6-epoxy-Chol, and 5,6-diOH-Chol have been employed for 5,6-epoxycholestan-3-ol, 5,6-epoxycholestan-3-ol, and cholestane-3,5,6-triol, respectively. In many recent articles in the biochemical and molecular biology literature, the important oxysterol (25R)-cholest-5-ene-3,26-diol [or (25R)-26-hydroxycholesterol] is referred to as 27-hydroxycholesterol. In this review, except in a few passages, I have used the more traditional nomenclature 26-hydroxycholesterol (26-OH-Chol), which is consistent with the systematic nomenclature utilized by Chemical Abstracts and the International Union of Pure and Applied Chemistry and does not imply a particular configuration at C-25 unless specified by the 25R- or 25S-designation. This nomenclature recognizes the fact that, in almost all biochemical studies, the configuration of C-25 in 26-hydroxysterols has not been established and, in those cases in which this matter has been studied in 26-hydroxysterols of biological origin, the concerned sterols are mixtures of the 25R- and 25S-isomers. 24,25-Epoxylanost-8-en-3-ol is designated as 24,25-epoxylanosterol, a name derived from the alkene (as in 2,3-epoxysqualene) rather than the alkane moiety (as in 24,25-epoxycholesterol). Many specialized abbreviations have been used and are identified in the text at their first use.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Chemical structure and numbering system for cholestane.

A number of general or specialized reviews of various aspects of research on oxygenated sterols have been published (56, 63, 80, 83, 93, 105, 187, 254, 303, 330, 331, 343, 358, 417, 442, 443, 465, 470, 469, 591, 768, 790, 792, 846, 876, 909, 911, 1000-1006, 1028, 1099, 1113, 1125).

	II. SEPARATION AND IDENTIFICATION OF OXYSTEROLS

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The isolation, characterization, and quantitation of oxysterols, as well as definition of their biosynthesis and metabolism, are critically dependent on methods for their separation. Presented below is an updating of current chromatographic methods (and their limitations) for the separation of various oxysterols and their derivatives. Almost all of the studies attempting to define the chromatographic properties of oxysterols have been limited by a lack of a truly comprehensive collection of authentic compounds, a situation resulting from the lack of the commercial availability of many oxysterols and the considerable effort and/or skill required to prepare these compounds by chemical synthesis. Uncritical application of chromatographic methods, in the absence of knowledge of the behavior of a substantial number of standards of defined structure and purity, is unfortunately frequently encountered and does not provide a firm basis for the identification and/or quantitation of a given oxysterol. Also presented are brief sections on mass spectrometry, NMR, and X-ray crystal analyses of oxysterols. Even these rigorous methods of identification are usually dependent on obtaining purified samples by chromatography.

A.  Chromatography of Oxysterols

1.  Thin-layer chromatography

Thin-layer chromatography (TLC) provides a very simple, rapid separation method. This approach has been extensively used in various studies of oxysterols. Useful chromatographic data have been provided by a number of investigators (21, 22, 44, 133, 360, 609, 844, 1108). Thin-layer chromatography can be very valuable in separation of certain mixtures, especially those from synthetic reaction mixtures, composed of relatively few and often predictable components. However, studies such as those of Aringer et al. (21) and Aringer and Nordström (22), in which the TLC behavior of a significant number of oxygenated sterol standards was evaluated (21, 22), clearly demonstrate the very limited capability of TLC to provide useful separations of oxygenated sterols that could be anticipated in complex mixtures as encountered in samples such as blood or tissues.

Brooks et al. (133) presented TLC data for the following oxysterols and their TMS ether derivatives: (24S)-24-OH-Chol, 26-OH-Chol, 7-OH-Chol, 7-OH-Chol, 25-OH-Chol, and (20S)-20-OH-Chol. Rennert et al. (844) reported TLC data for a limited number of oxygenated derivatives on Whatman HP silica plates (in order of increasing R_f): 7-OH-Chol, 4-OH-Chol, 26-OH-Chol, 25-OH-Chol, (22S)-22-OH-Chol, (22R)-22-OH-Chol, and 20-OH-Chol. Björkhem et al. (87) reported the TLC separation of the following oxysterols (in order of decreasing polarity): 7,12-dihydroxycholesterol, 5-cholestane-3,7,12-triol, 7-OH-Chol, 7-OH-Chol, 7-keto-Chol, 26-OH-Chol, and 7-hydroxycholest-4-en-3-one. Teng and Smith (1108) presented TLC behavior of a number of oxygenated derivatives of Chol and their ⁴-3-keto derivatives obtained after incubation of the individual oxysterols with Chol oxidase. Parent oxysterols included 7-OH-Chol, 7-OH-Chol, 19-OH-Chol, (20S)-20-OH-Chol, 25-OH-Chol, and (25R)-26-OH-Chol. The authors noted that 7-keto-Chol was unaltered upon incubation with cholesterol oxidase. Malavasi et al. (609) reported the TLC behavior of Chol, 7-hydroperoxycholest-5-en-3-ol (Chol 7-hydroperoxide), 7-keto-Chol, 7-OH-Chol, and 7-OH-Chol on silica gel F254 plates. Bachowski et al. (44) reported the TLC behavior on silica gel G-60 F254 plates for (in order of decreasing R_f) Chol, 5-hydroperoxycholest-6-en-3-ol, 7-hydroperoxycholest-5-en-3-ol, 7-hydroperoxycholest-5-en-3-ol, 7-keto-Chol, 5-cholest-6-ene-3,5-diol, 7-OH-Chol, and 7-OH-Chol. The studies of Aringer et al. (21) and Aringer and Nordström (22) provided data for a large number of oxygenated sterols, including those with oxygen functions at carbon atoms 3, 6, 7, 12, 15, 24, 25, and 26, with ⁵ or ⁴ olefinic bonds or a saturated sterol nucleus (with 5- and 5-isomers), and 5,6- and 4,5-epoxysterols. Even this relatively large collection of C-27 oxysterols did not include physiologically relevant oxysterols with oxygen functions at carbon atoms 4, 20, and 22 and the 24,25-epoxysterols. Aringer et al. (21) also presented data for a number of oxygenated derivatives of plant sterols. Gray et al. (348) reported the separation of the TMS derivatives of 25-OH-Chol and 5-cholestane-3,5-diol on silica gel G-AgNO₃ plates.

Liquid chromatography (LC), medium-pressure LC (MPLC), and HPLC on various supports have been utilized extensively in attempts to separate various oxygenated sterols. Retention data for a limited number of oxysterols on columns of Sephadex LH-20 and hydroxyalkylated Sephadex LH-20 have been presented (20-22). MPLC on silica gel (521) provided separation of the acetate derivatives of a number of C-27 oxysterols. However, the 5,6- and 5,6-isomers of 3-acetoxy-5,6-epoxycholestane were incompletely separated, and no separation of the diacetate derivatives of 7-OH-Chol and 7-OH-Chol was observed. MPLC on alumina-AgNO₃ provided complete separations of the acetate derivatives of 7-OH-Chol and 7-OH-Chol and of 5,6-epoxy-Chol and 5,6-epoxy-Chol (521). These striking separations, coupled with the relatively high capacity of this form of chromatography, should be of value in semi-preparative scale work involving the chemical syntheses of the concerned compounds.

Saucier et al. (899) presented HPLC retention time data for a limited number of oxysterols on normal and reverse-phase columns. Oxysterols studied (in order of elution on normal phase HPLC) were (24S)-24-OH-Chol, 7-keto-Chol, 25-OH-Chol, (25R)-26-OH-Chol, 7-OH-Chol, and 7-OH-Chol. The order of elution on the reverse-phase HPLC column was 7-OH-Chol, 7-OH-Chol, 24-OH-Chol, 7-keto-Chol, 25-OH-Chol, and 26-OH-Chol. Kudo et al. (521) presented normal phase and reverse-phase retention time data for the acetate derivatives of a number of oxygenated sterols including the following: 7-OH-Chol, 7-OH-Chol, 7-keto-Chol, 19-OH-Chol, (20S)-20-OH-Chol, (20S)-21-OH-Chol, (22R)-22-OH-Chol, (24S)-24-OH-Chol, 25-OH-Chol, (25R)-26-OH-Chol, 5,6-diOH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, (24RS)-24,25-epoxy-Chol, and (24RS)-24,25-epoxylanosterol. The use of acetate derivatives provided the opportunity, with the use of ¹⁴C- or ³H-labeled acetic anhydride, to follow the elution of the various compounds and, with knowledge of the specific activity of the labeled acetic anhydride, to quantitate the various oxysterols. Kermasha et al. (482) presented retention data for a limited number of oxygenated derivatives of Chol on normal-phase HPLC. Oxysterols included were (in order of elution) 20-OH-Chol, 25-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 7-keto-Chol, 7-OH-Chol, 7-OH-Chol, and 5,6-diOH-Chol. Caboni et al. (151) reported separations of a limited number of oxysterols by normal-phase HPLC. Oxysterols studied (in order of elution) were 5,6-epoxy-Chol, 5,6-epoxy-Chol (not separated from 4-OH-Chol), 20-OH-Chol, 7-keto-Chol, 25-OH-Chol, 19-OH-Chol, 7-OH-Chol, 7-OH-Chol, 7-hydroperoxycholest-5-en-3-ol, 7-hydroperoxycholest-5-en-3-ol, and 5,6-diOH-Chol. Brown et al. (135) reported retention data for a number of oxysterols on normal-phase HPLC on a silica column. Oxysterols studied included the following (in order of elution): 26-OH-Chol, 7-hydroperoxycholest-5-en-3-ol, 7-keto-Chol, 7-hydroperoxycholest-5-en-3-ol, 19-OH-Chol, 7-OH-Chol, and 7-OH-Chol. Lacritz and Jones (539) reported the use of normal-phase HPLC on an alumina-silica (16:84) column with gradient elution to separate a number of oxysterols. Included were (in order of elution) 20-OH-Chol, 7-keto-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 3-hydroxy-5-cholestan-6-one, 7-OH-Chol, and 7-OH-Chol. This work employed an evaporative light-scattering detector.

Ansari and Smith (13) presented data on the HPLC behavior of several oxysterols on a µPorasil column using hexane-isopropyl alcohol (24:1) as solvent and on a µBondapak C₁₈ column with acetonitrile-water (9:1). Data were presented on the following sterols: Chol, cholesta-3,5-dien-7-one, cholesta-4,6-dien-3-one, cholest-5-en-3-one, cholest-4-en-3-one, cholest-4-ene-3,6-dione, 25-OH-Chol, 7-keto-Chol, 5-hydroperoxy-5-cholest-6-en-3-ol, 7-hydroperoxycholest-5-en-3-ol, 7-hydroperoxycholest-5-en-3-ol, 5-cholest-6-ene-3,5-diol, 7-OH-Chol, and 7-OH-Chol. On the µPorasil column, little or no separation of cholest-5-en-3-one from cholesta-3,5-dien-7-one and cholesta-4,6-dien-3-one from cholest-4-en-3-one was observed. Little separation of numerous pairs of the oxysterols was observed on the µBondapak C₁₈ column (including 7-OH-Chol and 7-OH-Chol). Teng and Smith (1108) reported normal-phase HPLC retention times for a number of oxysterols that included (in order of elution) (20S)-20-OH-Chol, (24S)-24-OH-Chol, 23-OH-Chol, 25-OH-Chol, (25R)-26-OH-Chol, 19-OH-Chol, 7-keto-Chol, 7-OH-Chol, and 7-OH-Chol. Also presented were data for the corresponding ⁴-3-ketosterols formed upon incubation of the oxysterols with Chol oxidase. Sevanian and McLeod (952) presented data for normal-phase HPLC of the following sterols (in order of elution): 25-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 7-keto-Chol, 7-hydroperoxycholest-5-en-3-ol, and 7-OH-Chol. Lund et al. (592) presented retention data for reverse-phase HPLC of the following oxysterols (in order of elution): 24-OH-Chol, 25-OH-Chol, and 26-OH-Chol.

The HPLC data on hydroperoxide derivatives of Chol have been presented (13, 43, 506). Korytowski et al. (506) reported partial separations, by reverse-phase HPLC on a C₈ Ultrasphere column, of the following hydroperoxides (in order of elution): 7-hydroperoxycholest-5-en-3-ol, 7-hydroperoxycholest-5-en-3-ol, 5-hydroperoxycholest-6-en-3-ol, and 6-hydroperoxycholest-5-en-3-ol. Also reported was the normal-phase HPLC separation of the following hydroperoxides (in order of elution) on a silica column: 6-hydroperoxycholest-4-en-3-ol, 5-hydroperoxycholest-6-en-3-ol, and a mixture (not resolved) of the 7- and 7-hydroperoxides of Chol. Bachowski et al. (43) reported on the reverse-phase HPLC behavior of hydroperoxides of Chol on a C₁₈ Ultrasphere column. Under the conditions studied, the 7- and 7-hydroperoxides of Chol eluted together, and they were followed by 5-hydroperoxy-5-cholest-6-en-3-ol and 6-hydroperoxycholest-4-en-3-ol.

Saucier et al. (898) presented normal- and reverse-phase HPLC data for lanost-8-ene-3,32-diol and 3-hydroxylanost-8-en-32-al and for two isolated sterols believed to be lanosta-8,24-diene-3,32-diol and 3-hydroxylanosta-8,24-dien-32-al. Shiao et al. (968) reported reverse-phase HPLC data on oxygenated triterpenoids [specifically oxygenated derivatives of ^7,9(11),24-lanosta-trien-26-oic acid].

First applied in the sterol field for the separation of sterols differing in the number and location of olefinic double bonds (869, 870), Ag⁺-HPLC has been found to be valuable for the separation of the 3-acetate derivatives of (20R,22R)- and (20S,22S)-isomers of 20,22-di-OH-Chol (872).

Brooks et al. (131) reported gas chromatography (GC) retention data for the TMS ether derivatives of (in order of elution) 24-keto-Chol, 7-keto-Chol, 24-OH-Chol, and 25-OH-Chol on an SE-30 column. Gustafsson and Sjövall (368) presented GC data for the TMS ethers of (22R)-22-OH-Chol, (22S)-22-OH-Chol, 24-OH-Chol, and 26-OH-Chol on SE-30 and QF-1 columns. Under the conditions studied, little or no separation of the isomers of 22-OH-Chol was observed. Brooks et al. (133) provided retention data for the TMS ether derivatives of the following oxysterols on SE-30, OV-17, and QF-1 columns: 7-OH-Chol, 7-OH-Chol, (20S)-20-OH-Chol, (24S)-24-OH-Chol, 25-OH-Chol, and 26-OH-Chol. Burstein et al. (148) reported GC data for the TMS ether derivatives of (22R)-22-OH-Chol and (20R,22R)-diOH-Chol on an OV-1 column. Gray et al. (348) presented data for the mono-TMS and bis-TMS ether derivatives of 5-cholestane-3,5-diol, the bis-TMS ether derivative of 25-OH-Chol, and the TMS ether derivative of 5,6-epoxy-Chol on OV-1 and OV-17 columns. Also presented were data for the underivatized sterols. Aringer and Eneroth (20) reported GC retention data on the mono-, bis-, and tris-TMS ether derivatives of 5,6-diOH-Chol on SE-30 and QF-1 columns. Also presented were data for the bis- and tris-TMS derivatives of 5,6-diOH-Chol on an SE-30 column as well as data for 5,6-epoxy-Chol and 5,6-epoxy-Chol (and the corresponding epoxides of the 24-ethyl substituted sterols) as the free sterol and TMS and acetate derivatives. Aringer et al. (21) reported retention data on an SP-2100 column for the TMS derivatives of a number of oxygenated sterols, including (in order of elution) 7-OH-Chol, 7-OH-Chol, 5,6-epoxy-Chol (same retention time as 7-OH-Chol), (22R)-22-OH-Chol, 20-OH-Chol, 7-keto-Chol, 24-OH-Chol, 25-OH-Chol, and (25R)-26-OH-Chol. Also presented were data for a number of oxygenated derivatives of several plant sterols.

Aringer and Nordström (22) provided GC retention data for the TMS ether derivatives of a large number of dioxygenated C-27 sterols on an SP-2100 column (with additional data on a QF-1 column for some of the compounds). The various oxysterols included those with oxygen functions at carbon atoms 3, 6, 7, 12, 15, 24, 25, and 26, with ⁵- or ⁴-olefinic bonds or a saturated sterol nucleus (with 5- and 5-isomers), and 5,6- and 4,5-epoxysterols. Other potentially important oxysterols with oxygen functions at carbon atoms 4, 20, and 22 and 24,25-epoxysterols were not studied, nor were oxygenated derivatives of plant sterols. The data of Aringer and Nordström indicate that reliance on GC retention time data alone does not provide firm evidence for assignment of structure to an unknown oxysterol from a biological source in which considerable complexity can be anticipated. Brooks et al. (129) used selective enzymatic oxidation of the 3-hydroxyl groups of oxysterols with Chol oxidase to generate 3-ketosterols that were then analyzed by GC-MS of TMS derivatives. Kraaipoel et al. (511) reported the separation of the TMS ether derivatives of (in order of elution) (22R)-22-OH-Chol, (20S)-20-OH-Chol, (20R,22S)-20,22-diOH-Chol, and (20R,22R)-20,22-diOH-Chol on a capillary column coated with SE-30. Gumulka et al. (360) reported the capillary GC separations of 5,6-epoxy-Chol and 5,6-epoxy-Chol as the free sterols and as their acetate derivatives on SE-30 and SE-54 columns. Also presented were data for the TMS derivatives on the SE-54 column. Brooks et al. (132) presented retention data for the TMS derivatives of 5,6-epoxy-Chol, 5,6-epoxy-Chol, 7-keto-Chol, and 26-OH-Chol on DB-1 and DB-5 capillary columns.

Park and Addis (772) presented capillary GC data (DB-5 column) for the TMS derivatives of the following sterols (in order of elution): 7-OH-Chol, 7-OH-Chol, 4-OH-Chol, 5,6-epoxy-Chol, 3-hydroxy-5-cholestan-6-one, 7-keto-Chol, 25-OH-Chol, and 5,6-diOH-Chol. Under the conditions studied, the latter two sterols differed very little in retention time as was the case for the 4-hydroxy and 5,6-epoxy sterols. On a DB-1 column, the TMS ethers showed the following order of elution: 7-OH-Chol, 5,6-epoxy-Chol, 7-OH-Chol, 4-OH-Chol, 5,6-diOH-Chol, 7-keto-Chol, and 25-OH-Chol. Under the conditions studied, the retention times of the 7-hydroxy and 4-hydroxy sterols were very similar. Koopman et al. (505) presented capillary GC data for the TMS derivatives of a limited number of oxysterols on a fused silica capillary column coated with CP-Sil-19-CB. The sterols studied (in order of elution) were 7-OH-Chol, Chol, 19-OH-Chol, 7-OH-Chol, (22S)-22-OH-Chol, (22R)-22-OH-Chol, 20-OH-Chol, 25-OH-Chol, and 26-OH-Chol. Schmarr et al. (906) reported the capillary GC behavior of the TMS derivatives of a number of oxysterols on a modified 50% phenyl-50% methyl polysiloxane deactivated fused silica column. The following order of elution of the sterols was reported: 7-OH-Chol, 19-OH-Chol, Chol (eluted immediately after the 19-hydroxysterol and incompletely separated from it), 7-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 5,6-diOH-Chol, 25-OH-Chol, 20-OH-Chol, 7-keto-Chol, and 3,5-dihydroxy-5-cholestan-6-one. Rennert et al. (844) presented data for a limited number of oxysterols on capillary GC on a DB-17 column (as free sterols). The oxysterols studied and their retention times (relative to cholesterol) were 20-OH-Chol (1.35), 7-OH-Chol (1.40), 25-OH-Chol (1.41), (22R)-22-OH-Chol (1.44), (22S)-22-OH-Chol (1.44), (24RS)-24-OH-Chol (1.46), and 26-OH-Chol (1.63). Pizzoferrato et al. (811) reported baseline separations of the TMS ether derivatives of the following sterols (in order of elution) on an HP-1 cross-linked methyl silicone capillary column: 19-OH-Chol, 7-OH-Chol, 5,6-epoxy-Chol, 20-OH-Chol, 5,6-diOH-Chol, 7-keto-Chol, and 25-OH-Chol. Breuer (121) reported the capillary GC (HP Ultra-1 fused silica column) behavior of the TMS derivatives of the following oxysterols (in order of elution): 7-OH-Chol, 7-OH-Chol, 4-OH-Chol, 4-OH-Chol, 24-OH-Chol, 25-OH-Chol, and 26-OH-Chol. Lai et al. (538) presented data on the capillary GC behavior of the TMS ether derivatives of the following oxysterols (in order of elution) on a DB-1 column: Chol, 7-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 7-OH-Chol, 20-OH-Chol, 25-OH-Chol, 6-ketocholestanol, 7-keto-Chol, and 5,6-diOH-Chol.

Breuer and Björkhem (123) reported capillary GC (HP Ultra-1 fused silica column) data for the tert-butyldimethylsilyl ether derivatives of the following oxysterols (in order of elution): 5,6-epoxy-Chol, 7-keto-Chol, 7-OH-Chol, 4-OH-Chol, 24-OH-Chol, and 26-OH-Chol. Breuer et al. (124) reported that the TMS ether derivative of cholestane-3,5,6-triol eluted shortly after the TMS ether derivative of the corresponding 3,5,6-triol and shortly before that of 24-OH-Chol on an HP Ultra-1 fused silica capillary column. Breuer et al. (124) described a nice separation of the tert-butyldimethylsilyl ether derivatives of 4-OH-Chol and 4-OH-Chol. As noted above, Breuer (121) indicated that the TMS ethers of 4-OH-Chol and 4-OH-Chol are also separable on the same column. Mori et al. (673) presented capillary GC data on the TMS derivatives of the following oxysterols on an HP-1 cross-linked methyl silicone column: 7-OH-Chol, 5,6-epoxy-Chol, 4-OH-Chol, 5,6-diOH-Chol, 7-keto-Chol, and 25-OH-Chol. Pie et al. (801) and Pie and Seillan (802) reported essentially baseline separations of the TMS ether derivatives of the following compounds (in order of elution) on a 30-µm DB-5 capillary column: 7-OH-Chol, Chol, 19-OH-Chol, cholesta-3,5-dien-7-one, 7-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 20-OH-Chol, 5,6-diOH-Chol, 25-OH-Chol, 7-keto-Chol, and 26-OH-Chol. Garcia Regueiro and Maraschiello (322) reported baseline separations of the TMS derivatives of the following oxysterols (in order of elution) on a fused silica column coated with 5% phenylmethylsilicone: 7-OH-Chol, Chol, 19-OH-Chol, 7-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 20-OH-Chol, 5,6-diOH-Chol, 25-OH-Chol, and 7-keto-Chol.

The thermal instability of the hydroperoxides of Chol precludes their analyses by GC. Lercker et al. (558) studied the thermal decomposition of the 7- and 7-hydroperoxides of Chol acetate. Each of the hydroperoxides gave the corresponding 7-keto and 7- and 7-hydroxy analogs as the major products of thermal decomposition. Considerably lower levels of other products were reported and included materials believed to be cholesta-3,5,7-triene, cholesta-3,5-dien-7-one, Chol acetate, 7-dehydrocholesteryl acetate, 3-acetoxy-5,6-epoxycholestan-7-ol, 3-acetoxy-5,6-epoxycholestan-7-ol, and 3-acetoxy-5,6-epoxycholestan-7-ol. The GC data on the TMS derivatives of the various decomposition products were presented.

Brown et al. (134) reported retention data for the TMS ether derivatives of a selected group of oxysterols on a 30-m fused silica column coated with DB-5 MS [(5% phenyl)methylpolysiloxane]. The reported order of elution of the sterols was as follows: 7-OH-Chol, Chol, cholest-4-ene-3,6-diol, 19-OH-Chol, 7-OH-Chol, cholesta-3,5-dien-7-one, cholestane-3,5-diol, 5,6-epoxy-Chol, cholest-4-en-3-one (with no separation from the 5,6-epoxide), 6-hydroxycholest-4-en-3-one, (22R)-22-OH-Chol, 5,6-epoxy-Chol, 20-OH-Chol, 5,6-diOH-Chol, 25-OH-Chol, 3-hydroxy-5-cholestan-6-one, 7-keto-Chol, (25R)-26-OH-Chol, (25S)-26-OH-Chol (with no separation from its 25R-isomer). Axelson and Larsson (33) presented capillary retention data for the TMS derivatives of a number of oxysterols and the TMS derivatives of the methyl esters of several cholestenoic acid derivatives on a fused silica column (25 m × 0.32 mm) coated with a 0.17-µm layer of cross-linked methyl silicone with temperature programming. The sterols studied (in order of elution) were 7-OH-Chol, 7-hydroxycholest-4-en-3-one, 7-OH-Chol, 7-keto-Chol, 24-OH-Chol, 7,25-dihydroxycholesterol, 25-OH-Chol, 7,25-dihydroxycholesterol, 3-hydroxycholest-5-en-26-oic acid methyl ester, 7,26-dihydroxycholesterol, 26-OH-Chol, 7,25-dihydroxycholest-4-en-3-one, 7-hydroxy-3-oxo-cholest-4-en-26-oic acid methyl ester, 3,7-dihydroxycholest-5-en-26-oic acid methyl ester, 7,26-dihydroxycholest-4-en-3-one, 7,26-dihydroxycholesterol, 3-hydroxy-7-oxo-cholest-5-en-26-oic acid methyl ester, and 26-hydroxy-7-oxo-cholesterol.

Gustafsson and Sjövall (368) reported the separation of the 22R- and 22S-isomers of 22-OH-Chol, as their TMS derivatives, by GC on an SE-30 column. No separation was observed on a QF-1 column. Burrows et al. (145) reported separation of the 22S- and 22R-isomers of 22-OH-Chol by column chromatography and TLC of their 3-benzoate derivatives. Rennert et al. (844) reported no differences in the retention times of the 22R- and 22S-isomers of 22-OH-Chol upon capillary GC of the free sterols on a DB-17 column. The 22R- and 22S-isomers can be nicely separated by partition chromatography on columns of Celite 545 (149). The 23R- and 23S-isomers of 23-OH-Chol have been reported to have been cleanly separated by simple TLC of their dibenzoate derivatives (1159) and, as the free sterols, by silica gel column chromatography or by TLC (1158, 1159) but not by GC as the free sterols or as their diacetate derivatives on SE-30 or QF-1 columns (1158). As noted previously, the 20R,22R- and 20S,22S-isomers of 20,22-dihydroxycholesterol have been separated, as their 3-acetate derivatives, by Ag⁺-HPLC (872). 24R- and 24S-24-OH-Chol, as their TMS derivatives, can be resolved by capillary GC (592, 596). The 24R- and 24S-isomers of 24-OH-Chol can be separated with difficulty by TLC in the form of their 3-benzoate derivatives (856, 1039, 1159) or more readily by HPLC of the 3,24-dibenzoate derivatives (501). The 24R- and 24S-isomers of 24-OH-Chol can also be resolved, as their diacetate derivatives, by reverse-phase HPLC (521). The same sterols can also be nicely separated without derivatization by HPLC using a chiral column (899).

The 25R- and 25S-isomers of 26-OH-Chol or appropriate derivatives have been reported to be separable by HPLC (24, 839, 840, 1146). Redel and Capillon (840) reported the separation of the 3,26-diacetate derivatives of 25R- and 25S-26-OH-Chol ("at a 25-mg level") using 2 µPorasil columns (30 cm × 7.9 mm) using 2.5% ethyl acetate in hexane with 10 recycles. Uomori et al. (1146) reported a partial separation of the 25S- and 25R-isomers of 26-OH-Chol by HPLC on a TSK gel ODS-120T column (250 × 4 mm I.D.) using 7% water in methanol.

The 5,6- and 5,6-isomers of 5,6-epoxy-Chol can be separated (at times considerably less than completely) by GC in the form of the free sterols (360), their TMS ether derivatives (20, 360, 538, 772), and, more cleanly, as their acetate derivatives (22, 360). Simple TLC on silica gel plates provides little separation of the two compounds (20, 360). However, notable separations have been achieved by TLC on silica gel-AgNO₃ plates in the form of their acetate derivatives (20) or by TLC of their TMS derivatives on silica gel G plates pretreated with hexamethyldisilazane (20). Although only very slight separation of the acetates of the 5,6- and 5,6-epoxides was achieved on silica gel MPLC (521), remarkable separation of the two compounds has been effected by MPLC on an alumina-AgNO₃ column (521). Excellent separations of the acetate or benzoate derivatives of the two epoxides can be achieved by both normal-phase and reverse-phase HPLC (13, 521). Sevanian and McLeod (952) also reported separation of the 5,6-epoxide isomers as the free sterols by normal-phase HPLC. Cholestane-3,5,6-triol and cholestane-3,5,6-triol can be separated by TLC, LC, or GC (as either di-TMS or tri-TMS derivatives) (20).

The 24R- and 24S-isomers of 24,25-epoxy-Chol and of 24,25-epoxylanosterol are resistant to separation by capillary GC as their acetate derivatives (280) or by reverse-phase HPLC on routine C₁₈ columns (280, 758, 759) either as the free sterols or their acetate derivatives. However, these isomers are separable on a special Vydac C₁₈ column (280, 759). The benzoates of the 24R- and 24S-isomers of 24,25-epoxy-Chol have been separated by normal-phase HPLC (280, 946), by MPLC on silica gel 60 (897), and by preparative TLC (701). The separation of the 24R- and 24S-isomers of 24,25-epoxylanosterol by TLC of the acetate or benzoate derivatives has also been described (1102, 1103). The free sterols have been reported to show slightly different retention times on normal-phase HPLC (1102), with retention times of 16.6 and 16.9 min for the 24S- and 24R-isomers, respectively.

Oxygenated sterols are often found as their fatty acid esters. Reverse-phase HPLC procedures for the separation of fatty acid esters of 3-hydroxy-5-cholest-8(14)-en-15-one have been described (207). With the use of chemically synthesized (825) fatty acid esters of the 15-ketosterol, rather clean separations of the linolenate, arachidonate, linoleate, elaidate, oleate, and stearate esters were achieved using a C₁₈ Microsorb column. With the use of this system, separation of the oleate and palmitate esters was not achieved. However, the use of a C₆ Spherisorb column permitted the separation of the linoleate, arachidonate, linoleate, palmitate, oleate, stearate, and arachidate esters of the 15-ketosterol. Lin and Morel (571) reported TLC and reverse-phase HPLC data for the mono- and dioleate esters of 25-OH-Chol. Brown et al. (134) described reverse-phase HPLC separations of the palmitate, stearate, oleate, linoleate, arachidonate, and docosahexaenoate esters of 7-keto-Chol. The individual esters were prepared by chemical synthesis, but they were not isolated in pure form (except for the oleate ester) and were not characterized. Szedlacsek et al. (1082) presented retention time data for a number of diesters and 3-acyl monoesters of (25R)-26-OH-Chol on an Adsorbosphere C₁₈ column using mixtures of acetonitrile and isopropyl alcohol as the eluting solvent. Also presented were data for the 3-oleoyl esters of 7-OH-Chol, 7-OH-Chol, 7-keto-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 5,6-diOH-Chol, and 25-OH-Chol.

Full or partial MS data for a number of oxygenated derivatives of Chol and/or their TMS derivatives have been presented. These include 26-OH-Chol (21, 32), 25-OH-Chol (131, 155, 772, 811), 24-OH-Chol (131, 368), 22-OH-Chol (148, 368, 1063), 20-OH-Chol (811), 19-OH-Chol (562), 7-OH-Chol (562, 772), 7-OH-Chol (155, 562, 772, 811), 4-OH-Chol (124), 4-OH-Chol (124, 562, 772), 5,6-diOH-Chol (124, 562, 772, 811), 5,6-diOH-Chol (124), 5,6-epoxy-Chol (74, 348, 562, 772, 811, 155), 5,6-epoxy-Chol (20, 562), 7-keto-Chol (131, 155, 562, 772, 811), 24-keto-Chol (131), cholestane-3,5-diol (155), 7,26-diOH-Chol (1222), 7,26-dihydroxycholest-4-en-3-one (562), 7,26-dihydroxycholest-4-en-3-one (562), (20R,22R)-20,22-diOH-Chol (148, 511, 872, 1063), (20S,22S)-20,22-diOH-Chol (872), and cholesta-3,5-dien-7-one (74). In addition, Aringer and Nordström (22) presented fragmentation data for the TMS derivatives of a large number of oxygenated sterols. These included the mono-TMS ether derivatives of a number of 3-hydroxysterols with ketone functions at carbon atoms 6, 7, 12, 15, and 24, the TMS ether derivatives of 3-hydroxysterols with either 5,6- or 4,5-epoxide functions, and bis-TMS ether derivatives of 3-hydroxysterols with an additional hydroxy function at carbon atoms 6, 7, 12, 15, 24, 25, or 26. Partial mass spectral data for a number of oxygenated derivatives of -sitosterol have been presented by Daly et al. (232). MS of the TMS ether derivatives of a number of oxygenated derivatives of campesterol and sitosterol were presented by Dutta and Appelqvist (270). Dutta (269) presented mass spectra for several oxygenated derivatives of stigmasterol. However, the oxidized derivatives of these plant sterols were not isolated and fully characterized prior to GC-MS analyses.

Considerable effort has been expended toward an understanding of the electron impact-induced fragmentations of 3-hydroxy-5-cholest-8(14)-en-15-one. Studies of the 15-ketosterol and its derivatives (including its TMS dienol ethers) and analogs (including deuterium-labeled analogs) along with analyses of high-resolution MS data on individual fragment ions provided important information regarding its fragmentation on electron impact (825, 827, 828) which have facilitated studies of the structures of metabolites (485, 826, 827, 916) and new analogs (382-384, 486, 978-983, 1058, 1071, 1072, 1074-1076, 1201) of the parent 15-ketosterol. MS data have been presented for a number of chemically synthesized fatty acid esters of 3-hydroxy-5-cholest-8(14)-en-15-one (825). Included were data for the palmitate, palmitoleate, stearate, oleate, linoleate, linolenate, arachidate, and arachidonate esters.

With continued improvements in instrumentation and techniques, NMR spectroscopy provides an increasingly powerful approach for the identification of oxygenated sterols. Selected aspects of the NMR analyses of oxygenated sterols have been reviewed elsewhere (1198). In efforts led by Dr. W. K. Wilson, the ¹H- and ¹³C-NMR spectral properties of 3-hydroxy-5-cholest-8(14)-en-15-one and its analogs have been very extensively studied and provided important evidence for the structures of metabolites and analogs of the 15-ketosterol (382, 384, 485, 486, 826, 827, 978-983, 1072-1074, 1076, 1129, 1130, 1198, 1201). In addition, they have provided critical information leading to the first assignments of the resonances for each of the protons of the side chain of sterols with a saturated C₈H₁₇ side chain (as in Chol) or of sterols with a ²⁴ double bond in the side chain (980, 1199).

Carr et al. (163) applied ¹H- and ¹³C-NMR to the study of 6-chloro-5-cholestane-3,5-diol, the major product obtained upon treatment of Chol-lecithin liposomes with hypochlorous acid. ¹³C-NMR assignments were provided for this halohydrin and for two other halohydrins of Chol, i.e., 6-chloro-5-cholestane-3,5-diol and 5-chloro-5-cholestane-3,6-diol. Partial ¹H-NMR assignments were provided for the 6-chloro-3,5-diol. Emmons et al. (279) presented detailed analyses of the ¹³C- and ¹H-NMR spectra of a number of oxygenated derivatives of lanosterol and 24,25-dihydrolanosterol. Emmons et al. (280) also presented full ¹³C-NMR assignments for the acetate derivatives of (24R)-24,25-epoxylanosterol, (24S)-24,25-epoxylanosterol, (24R)-24,25-epoxy-Chol, and (24S)-24,25-epoxy-Chol. The 24R- and 24S-isomers of the acetates of 24,25-epoxylanosterol and of 24,25-epoxy-Chol could be differentiated by ¹³C-NMR. In contrast, the ¹H-NMR spectra of the 24R- and 24S-isomers of the epoxylanosterol and epoxycholesterol were essentially indistinguishable, as had been noted previously for the acetates of 24,25-epoxylanosterol (100). The 24R- and 24S-isomers of 24-hydroxysterols can be differentiated by ¹³C-NMR (500). The 25R- and 25S-isomers of 26-hydroxysterols can be differentiated by ¹³C-NMR (54, 826, 949, 1146) and by ¹H-NMR of their (+) or ()-methoxy(trifluoromethyl)phenylacetate esters (711, 1146) or acetate esters (826).

A nice application of ¹H- and ¹³C-NMR to the elucidation of structure of a derivative of an oxysterol is found in studies of the structure of squalamine (674, 1192), a novel compound isolated from the shark that has notable antimicrobial activities. Another case is that of boophiline, a 3-sulfate derivative of 3-hydroxycholest-5-en-26-oic acid in which the acid is conjugated in an amide linkage to L-leucine (819). This novel compound, isolated from a cattle tick, was reported to have antifungal and antibacterial activity (819). Kobayashi (499) analyzed the ¹³C-NMR spectra of a number of polyhydroxy 5,14-steroids and determined additivity relationships of hydroxyl substituent effects. Fontana and co-workers (307, 308) applied ¹H-NMR in studies of the levels of certain oxygenated derivatives of Chol in egg powders. Their studies involved examination of the resonances corresponding to C-6 and C-18 protons of free oxysterol fractions obtained after chromatography. The detection limit under the conditions employed was ~0.3 ppm or 5 mg from a 16-g sample of egg powder. The oxygenated derivatives of Chol studied (307, 308) were the following: 5,6-epoxy-Chol, 5,6-epoxy-Chol, 7-OH-Chol, 7-OH-Chol, 7-keto-Chol, 5,6-diOH-Chol, 25-OH-Chol, and 20-OH-Chol. Although the spectra were recorded at 500 MHz, only low-precision data were presented, and the number of oxysterols studied was limited.

X-ray crystallographic analysis has been employed to establish unequivocally the structures of oxygenated sterols (or their derivatives) shown to be potent regulators of sterol synthesis (112, 337, 485, 665, 707, 767, 800, 922, 927, 1030, 1031) or of key intermediates or by-products in the chemical syntheses of 15-oxygenated sterols of potential importance in biology or medicine (214, 1195, 1196). X-ray crystallographic analyses of the 25R-isomer of 26-OH-Chol (493, 949) and its 25S-isomer (1146) have been presented. Duchamp et al. (262) reported X-ray data on the p-bromobenzoate ester of (25S)-26-hydroxycholest-4-en-3-one. McCourt et al. (629) provided X-ray crystal structure analyses for 25-OH-Chol and 7-keto-Chol. Ishida et al. (427) presented an X-ray crystal analysis of scymnol [(24R)-5-cholestane-3,7,12,24,26,27-hexol], a compound with reported medical utility from shark bile. Shimura et al. (974) reported the determination of the structure of aragusterol C by X-ray crystal analysis. Aragusterol C, a novel halogenated (chloro)oxysterol from an Okinawan sponge, was reported to inhibit the growth of KB cells (IC₅₀ 0.04 µg/ml) and had antitumor activity in vivo.

	III. FORMATION OF OXYSTEROLS

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In 1956, Fredrickson and Ono (314) reported the formation of labeled 26-OH-Chol and 25-OH-Chol upon incubation of [4-¹⁴C]Chol with mouse liver mitochondria. The labeled products were characterized by their mobilities on paper chromatography and, in the case of the 26-hydroxysterol, by cocrystallization with the authentic, unlabeled sterol. In 1961, Danielsson (234) also characterized 26-OH-Chol as a product of the mitochondrial metabolism of [4-¹⁴C]Chol. The labeled 26-hydroxysterol was characterized by chromatography and cocrystallization experiments. In contrast to the study of Fredrickson and Ono, little or no enzymatic formation of labeled 25-OH-Chol could be detected. Upon incubation of the labeled 26-OH-Chol with mouse liver homogenates, the formation of cholest-5-ene-3,7,26-triol was shown by chromatography and cocrystallization experiments. In a separate study, Danielsson (235) reported the formation of cholest-5-ene-3,7,26-triol after incubation of mouse liver homogenates with labeled 7-OH-Chol. Mitropoulos and Myant (659) reported the conversion of [4-¹⁴C]Chol to 26-OH-Chol, 3-hydroxycholest-5-en-26-oic acid, and 3-hydroxychol-5-en-24-oic acid (as well as lithocholic acid, chenodeoxycholic acid, and - and -muricholic acids) when incubated with rat liver mitochondria in the presence of the soluble fraction of a rat liver homogenate. For the most part, characterization of the labeled products was limited to TLC. The C-24 acids were reported to be present in the form of taurine conjugates. Mitropoulos et al. (660) reported the conversion of [4-¹⁴C]Chol to 26-OH-Chol upon incubation with rat liver mitochondria. The labeled 26-OH-Chol was characterized by TLC behavior of the free sterol and its diacetate derivative and by the results of cocrystallization studies with the diacetate derivative.

Björkhem and Gustafsson (90) reported the formation of 26-OH-Chol from Chol with rat liver mitochondria and the requirement for NADPH and oxygen. Incubation under nitrogen or incubation with buffer alone or boiled mitochondria was reported to result in no formation of 26-OH-Chol. The product was characterized by chromatography (TLC and radio-GC). The formation of the 26-hydroxysterol was shown to occur with the incorporation of molecular oxygen. Björkhem and Gustafsson (90) also observed the formation of 25-OH-Chol from Chol with rat liver mitochondria in the presence of NADPH and oxygen. The formation of the 25-hydroxysterol was very much less than that of 26-OH-Chol under the same conditions. Identification was based on TLC and GC. No formation of labeled 25-OH-Chol was detected upon incubation with buffer alone or with boiled mitochondria. Evidence was presented indicating the origin of the 25-hydroxyl function from molecular oxygen. Aringer et al. (21) reported the enzymatic formation of 26-OH-Chol, 25-OH-Chol, and 24-OH-Chol upon incubation of [4-¹⁴C]Chol with rat liver mitochondria in the presence of NADPH or an NADPH-generating system. The relative amounts of the 26-hydroxy-, 25-hydroxy-, and 24-hydroxysterols were ~1.0, 0.3-0.5, and 0.1, respectively. The formation of these sterols from autoxidation of Chol was excluded on the basis of results with boiled mitochondrial controls. Characterization of the oxysterols was based on TLC and LC and on GC-MS studies of their TMS derivatives.

Pedersen and Saarem (785) reported the solubilization of a cytochrome P-450 from rat liver mitochondria that catalyzed the conversion of Chol into 26-OH-Chol and 25-OH-Chol (in a ratio of 9:1) in the presence of adrenodoxin, NA