I. INTRODUCTION

Top Previous Next References

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

Top Previous Next References

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

Top Previous Next References

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, NADPH-ferredoxin reductase, and NADPH. The oxysterols were characterized by GC-MS as their TMS derivatives. Pedersen et al. (786) studied the substrate specificity of the soluble liver mitochondrial P-450 system. The most efficient substrates were 7-OH-Chol and 7-hydroxycholest-4-en-3-one followed by 5-cholestane-3,7,12-triol and 5-cholestane-3,7-diol. Chol was hydroxylated at a considerably lower rate and gave ~9:1 mixture of 26-OH-Chol and 25-OH-Chol. With the soluble mitochondrial preparation, 5-cholestane-3,7,12-triol gave 5-cholestane-3,7,12,26-tetrol as the major product and 3,7,12-trihydroxy-5-cholestan-26-oic acid as a minor product. Gustafsson (363) demonstrated the 26-hydroxylation of exogenous and endogenous Chol by rat liver mitochondria. The 26-hydroxysterol was characterized by GC-MF of its bis-TMS derivative. Kok and Javitt (502) reported the 26-hydroxylation of endogenous Chol of hamster liver mitochondria using GC-MS methodology involving a deuterated internal standard of 26-OH-Chol with analyses of ion abundances in the region corresponding to M-CH₃COOH in the MS of the diacetate derivative. The description of the characterization of the labeled internal standard was limited.

Lund et al. (593) reported the conversion of Chol to 26-OH-Chol and 24-OH-Chol upon incubation with mouse liver mitochondria in the presence of NADPH and isocitrate. No formation of 25-OH-Chol was detected under the conditions employed. The amounts of the 26-OH-Chol and the 24-OH-Chol formed were not presented (although the former compound was reported to be more abundant). On the basis of 1-h incubations with mouse liver mitochondria, the presence of an isotope effect in the conversion of d₆-Chol (deuterium at C-26 and C-27) to 26-OH-Chol and in the conversion of d₂-Chol (deuterium at C-24) to 24-OH-Chol was reported. Lund et al. (592) demonstrated the conversion of Chol to 26-OH-Chol, 25-OH-Chol, and 24-OH-Chol (1.0:0.2:0.04, respectively) upon incubation with pig liver mitochondria in the presence of added isocitrate in Tris·HCl buffer containing 20% glycerol. The products were identified by GC-MS of TMS derivatives. Incubation of a highly purified preparation of the 26-hydroxylase from pig liver mitochondria with [6,7,7-²H₃]Chol in the presence of adrenodoxin, adrenodoxin reductase, and NADPH gave not only 26-OH-Chol but also the 25-hydroxy and 24-hydroxy derivatives of Chol. The ratio of the 26-hydroxy, 25-hydroxy-, and 24-hydroxy derivatives of Chol was reported to be approximately the same as that observed with the intact pig liver mitochondria. The 24-OH-Chol formed with the purified enzyme was reported to be one isomer that was tentatively assigned as the 24S configuration. The authors suggested that a single enzyme was responsible for the formation of the three hydroxysterols. The presence of an isotope effect was reported for 24-hydroxylation with the purified enzyme (reported as "K_H/K_D >10") but little or none for 25-hydroxylation or 26-hydroxylation. The absence of an isotope effect for 26-hydroxylation with the purified pig liver enzyme differed from the finding of a significant isotope for the same reaction catalyzed by mouse liver mitochondria (593). The magnitude of isotope effects was estimated from the results of a single time point experiment (30 min), and no information regarding the amounts of oxygenated sterol products formed was provided. The deuterated substrates used contained deuterium not only at the carbon atom of interest but also at adjacent carbons. For the 26-, 25-, and 24-hydroxylations the substrates used were [25,26,26,26,27,27,27-²H₇]Chol, [25,26,26,26,27,27,27-²H₇]Chol, and [23,23,24,24,25-²H₅]Chol, respectively. The preparation of the 24-OH substrate was described in another paper by the same investigators (593). The product was obtained in low yield through a multistep synthesis. Little or no characterization of the product, or of the multiple intermediates in its synthesis, was provided. The final product was reported to contain 77.4% d₅ species accompanied by lower percentages of d₄, d₃, d₂, d₁, and d₀ species. In the critical experiment concerning the 24-hydroxylation, essentially no 24-hydroxylated species was formed from the [23,23,24,24,25-²H₅]Chol. In this experiment, the Chol substrate lacking deuterium at C-24 was a [25,26,26,26,27,27,27-²H₇]Chol preparation.

Pyrek et al. (826) demonstrated that incubation of 3-hydroxy-5-cholest-8(14)-en-15-one with rat liver mitochondria in the presence of NADPH gave the 25R- and 25S-isomers of the 26-hydroxy derivative of the 15-ketosterol as well as the 25-hydroxy and 24-hydroxy derivatives of the 15-ketosterol. The ratio of the 26-hydroxy, 25-hydroxy, and 24-hydroxy sterols was similar to that reported for the side-chain oxidation of Chol with rat liver mitochondria (21) and pig liver mitochondria (592). Both isomers of the 26-hydroxy-15-ketosterol were formed, with a ratio of 25R to 25S of ~4:1 as determined by ¹H- and ¹³C-NMR analyses.

Petrack and Lantario (798) described an assay method for 26-hydroxylase activity that involved normal-phase HPLC of the ⁴-3-ketones resulting from treatment of mitochondrial incubation mixtures with Chol oxidase. Products of the action of Chol oxidase on authentic oxysterols (7-OH-Chol, 7-OH-Chol, 26-OH-Chol, and 25-OH-Chol) were studied by normal-phase HPLC. Only an authentic sample of 7-hydroxycholest-4-en-3-one was described as being available. 24-OH-Chol was not studied. Products of the action of Chol oxidase on the authentic standards and on mitochondrial incubation mixtures were studied only by HPLC (absorbance at 240 nm). Using this assay system, the authors reported that rat liver mitochondria catalyzed the formation of 26-OH-Chol from endogenous Chol. No 26-OH-Chol was formed with boiled mitochondrial preparations or upon incubation in the presence of cyclosporin (20 µM) or carbon monoxide. The enzymatic formation of lesser amounts of 25-OH-Chol was noted. 2-Hydroxypropyl--cyclodextrin (0.9%) and exogenous Chol in -hydroxypropyl--cyclodextrin (0.9%) stimulated 26-OH-Chol formation. It was suggested that stimulation by cyclodextrin itself was due to facilitation of access of the endogenous Chol substrate to the enzyme. Fasting markedly lowered 7-OH-Chol formation, but not 26-OH-Chol formation, in homogenates of rat liver.

Since early studies by Berseus (69) and Mitropoulos and Myant (658), the stereospecificity involved in the formation of 26-hydroxy derivatives of Chol and of other sterols (27, 55, 367, 374, 963, 1144, 1145) has been an area of interest. The experiments of Berseus (69) and of Mitropoulos and Myant (658), both involving the use of [¹⁴C]Chol formed biosynthetically from [2-¹⁴C]mevalonate, demonstrated that the formation of 26-OH-Chol by mouse liver mitochondria involved a high degree of stereospecificity. The in vivo results of Hanson et al. (374), based on studies of the 3,7,12-trihydroxy-5-cholestan-26-oic acid formed from [2-¹⁴C]mevalonate, were similar and, in addition, indicated that the 26-oic acid had predominantly the 25R-configuration. Batta et al. (55) reported that the 3,7,12-trihydroxy-5-cholestan-26-oic acid of human bile had the 25R-configuration. However, Une et al. (1145) reported that the 3,7,12-trihydroxy-5-cholestan-26-oic acid found in the unconjugated fraction of urine from an infant with Zellweger's syndrome was, as judged by HPLC analysis of its p-bromophenacyl derivative, a 7:3 mixture of the 25R- and 25S-isomers. It should be noted that the efficient in vivo conversion of both the 25R- and 25S-isomers of 3,7,12-trihydroxy-5-cholestan-26-oic acid to cholic acid has been observed in the rat (126, 365) and human (1078). Very recently, Van Veldhoven et al. (1161) reported that purified trihydroxycoprostanoyl-CoA oxidase of rat liver, catalyzing the introduction of a ²⁴-double bond, acted on only the 25S-isomer of the CoA derivative of 3,7,12-trihydroxy-5-cholestan-26-oic acid. However, isolated rat liver peroxisomes catalyzed the desaturation of both the 25S- and 25R-isomers, leading to the suggestion (1161) of the presence of a racemase in the peroxisomes.

The stereospecificity of the -hydroxylation of sterols by mitochondria and by microsomes of liver has been reported to differ. In one study (963) with liver mitochondria and microsomes of the rat, guinea pig, and rabbit, incubation of 5-cholestane-3,7-diol was reported to yield both the 25R- and 25S-isomers of cholestane-3,7,26-triol. However, the 25R-isomer was the predominant product (as judged by GC of the TMS derivative) in the mitochondrial incubations. With liver microsomes of the rat and guinea pig, the 25S-isomer was the predominant product, whereas with rabbit, the 25R-isomer was reported to be the predominant product. The results with rat liver microsomes were in accord with an earlier report by Gustafsson and Sjöstedt (367) that indicated that the 3,7,12-trihydroxy-5-cholestan-26-oic acid formed upon incubation of biosynthetically labeled (from [2-¹⁴C]mevalonate) Chol with rat liver mitochondria had the 25S-configuration. Une et al. (1144) reported that, upon incubation of 5-cholestane-3,7,12-triol with liver mitochondria of a number of different animals (rat, rabbit, hamster, chicken, turtle, carp, and frog), both the 25R- and 25S-isomers of 5-cholestane-3,7,12,26-tetrol were formed. However, the 25R-isomer was the predominant product in each case (as judged by HPLC analysis of a 26-anthroyl derivative). Incubations of the triol with liver microsomes of hamster, chicken, frog, and carp were also reported to give both the 25R- and 25S-isomers of the tetrol. In accord with the reports of Shefer et al. (963) and Gustafsson and Sjöstedt (367), the 25S-isomer was the predominant product with rat liver microsomes. However, the 25R-isomer was the major product with hamster and carp liver microsomes. The conversion of 5-cholestane-3,7,12-triol to 5-cholestane-3,7,12,26-tetrol by a partially purified cytochrome P-450 from rat liver mitochondria was reported to give only the 25R-isomer of the tetrol (as judged by TLC) (27). However, the published chromatogram does not appear to exclude the possible formation of some of the 25S-isomer. As noted previously, the -hydroxylation of 3-hydroxy-5-cholest-8(14)-en-15-one by rat liver mitochondria has been shown to yield both the 25R- and 25S-isomers of 3,26-dihydroxy-5-cholest-8(14)-en-15-one. NMR studies indicated that the 25R- and 25S-isomers were present in a 4:1 ratio. A similar direct analysis of the stereochemistry of the in vitro enzymatic -hydroxylation of Chol has not been made. Whereas Chol serves as a substrate for the -hydroxylation system of mitochondria, it is not clear whether Chol is a substrate for the microsomal system (367). However, it is noteworthy that a sample of 26-OH-Chol isolated from human aorta has been reported (839) to be comprised of its 25R- and 25S-isomers in a 9:1 ratio (as judged by HPLC analysis of its diacetate derivative).

As noted previously, 24-hydroxysterols and 25-hydroxysterols can arise from mitochondrial hydroxylation of sterols as shown in the cases of Chol (21, 592) or 3-hydroxy-5-cholest-8(14)-en-15-one (826, 827). The formation of 24-OH-Chol has also been reported (251) after aerobic incubation of labeled Chol with a microsomal preparation of bovine brain in the presence of an NADPH-generating system or the combination of NADPH plus an NADPH-generating system. The characterization of the labeled product was based on TLC and cocrystallization experiments with the dibenzoate derivative. The results of the latter experiments were interpreted as indicating the 24S-configuration of the 24-hydroxysterol. The extent of conversion of Chol to 24-OH-Chol was very low (0.32-0.38%). No conversion was detected in an incubation carried out in the absence of oxygen. Similar results were obtained with rat brain microsomes (575), with conversions of Chol to 24-OH-Chol of ~0.15-0.18%. Björkhem et al. (94) reported that incubations of rat brain microsomes with [4-¹⁴C]Chol and NADPH gave negligible conversion to 24-OH-Chol, stated to be considerably <0.2%. The authors noted that "the small extent of conversion of the labeled cholesterol varied considerably in different sets of experiments, regardless of the time of incubation and the mode of addition of the cholesterol (acetone, cyclodextrin, or Tween 80)." Despite the difficulties in demonstration of the enzymatic formation of 24-OH-Chol using radiotracer methodology, the authors indicated the in vitro formation of the 24-hydroxysterol by two types of experiments. The first involved GC-MS measurement of the levels of 24-OH-Chol in rat brain microsomes before and after incubation for 2 or 20 h. The levels of the 24-hydroxysterol after 0, 2, and 20 h of incubation in a typical experiment were reported as 0.72, 0.82, and 0.86 µg/ml brain microsomal preparation. However, this approach apparently gave variable results, and it was stated that "in some experiments there was no linear increase in the amount of 24(S)-hydroxycholesterol." The second approach involved the measurement, by GC-MS, of the incorporation of labeled oxygen of ¹⁸O₂ into the 24-hydroxysterol formed upon incubation of rat brain microsomes in the presence of NADPH. The rate of formation of the 24-OH-Chol was estimated to be ~30 ng·h¹·ml microsomal preparation¹ or ~0.015% of the Chol in the preparation per hour. The mitochondrial fraction of rat brain also catalyzed the incorporation of labeled oxygen of ¹⁸O₂ into 24-OH-Chol. The level of incorporation (~0.003% of endogenous Chol per hour) was lower than that observed with the microsomal fraction.

24-Hydroxysterols can also result from another process, i.e., the reduction of a 24,25-epoxysterol. Steckbeck et al. (1039) reported the formation of labeled (24R)-24-hydroxylanost-8-en-3-ol and (24R)-24-OH-Chol upon incubation of [2-³H]-(24R),25-epoxylanosterol with a rat liver homogenate preparation. However, it should be noted that the 24R-isomer of 24-OH-Chol is not considered to be the naturally occurring isomer present in mammalian blood and tissues. In subsequent studies (1029, 1102), no conversion of ³H-labeled (24S),25-epoxy-Chol to 24-OH-Chol or 25-OH-Chol could be detected with mouse L fibroblasts. Similarly, with Chinese hamster lung cells, no conversion of (24S)-24,25-epoxy-Chol to 24-OH-Chol or 25-OH-Chol was detected. However, with these cells, conversion of the (24R)-24,25-epoxy-Chol to (24R)-24-OH-Chol was observed.

Saucier et al. (897) reported the formation of labeled 25-OH-Chol (identified by chromatographic and cocrystallization experiments) upon incubation of [³H]mevalonate with Chinese hamster lung cells. The mode of formation of the 25-OH-Chol was not established. Alsema et al. (7) reported experiments suggesting 25-hydroxylation of (20S)-20-OH-Chol by bovine adrenal cortex mitochondria. Formation of the 25-hydroxylated sterol was observed in incubations carried out at pH 7.80 (but not at pH 7.40). Assignment of structure was based on GC-MS studies of the TMS ether derivative (in the absence of an authentic standard).

Pedersen et al. (787) reported that a solubilized cytochrome P-450 from bovine brain mitochondria catalyzed the 26-hydroxylation of a number of C₂₇ sterols (5-cholestane-3,7,12-triol, 7-hydroxycholest-4-en-3-one, 7,12-dihydroxycholest-4-en-3-one, 5-cholestane-3,7-diol, and 5-cholestane-3,7,12-triol). The activity of the brain preparation was reported to be considerably lower than comparable preparations from rat and human liver. No formation of 26-OH-Chol from Chol was detected under the conditions studied. The products were characterized by GC-MS, although data on this matter were not presented. No mention was made regarding hydroxylation at C-24 or C-25 by the bovine brain enzyme preparation. Javitt et al. (448) reported the formation of labeled 26-OH-Chol (as well as Chol, 3-hydroxychol-5-en-26-oic acid, chenodeoxycholic acid, and cholic acid) after the intravenous administration of [5-¹³C]mevalonate to animals. The title of this paper indicates that the studies were made in the Syrian hamster, whereas the text indicates that the studies involved Sprague-Dawley rats.

Rennert et al. (844) demonstrated the expression of the 26-hydroxylase gene in human granulosa cells. They also reported the NADPH-dependent formation of labeled 26-OH-Chol from [³H]Chol (location of label not specified) upon incubation with mitochondria from ovarian mitochondria from superovulated rats. All incubations were carried out in the presence of aminoglutathimide (100 µg/ml) to inhibit side-chain cleavage activity that was reported to be much higher than that of 26-hydroxylase activity. Identification of the product was based on chromatographic and cocrystallization studies as well as GC-MS. However, it should be noted that at least one significant ion was present in the spectrum of the isolated sterol that was not present in that of authentic 26-hydroxysterol and that at least one major ion present in the MS of the authentic sample was absent in the MS of the isolated sterol. Ovarian mitochondrial 26-hydroxylase activity was markedly increased by calcium (200 µM), and it was inhibited by ketoconazole (IC₅₀ <10 µM), pregnenolone (IC₅₀ <10 µM), or progesterone (IC₅₀ ~10 µM). It was suggested that the levels of progestins that inhibit the 26-hydroxylase may be in the physiological range for their occurrence in ovaries. The authors also noted that physiological stimulation of steroidogenesis in ovary might, by virtue of the actions of progestins on the 26-hydroxylase, suppress the formation of 26-OH-Chol. Furthermore, such a reduction in 26-OH-Chol formation could result in increased Chol synthesis and increased LDL receptor activity, with a resulting continual supply of Chol for cell function and steroidogenesis.

Su et al. (1057) characterized a cDNA for a mitochondrial P-450 of rat liver and expressed it in COS cells. Mitochondrial preparations from the COS cells were reported to catalyze, in reconstituted systems, the 25-hydroxylation of vitamin D₃ and the 26-hydroxylation of Chol. Characterization of the products was limited to HPLC analysis. Usui et al. (1147) presented evidence indicating that the vitamin D 25-hydroxylase of rat liver mitochondria also catalyzed the 26-hydroxylation of 5-cholestane-3,7,12-triol. The cDNA for the vitamin D 25-hydroxylase was transfected into COS cells, and the enzyme activities of a solubilized extract of mitochondria from the COS cells were studied. A reconstituted system containing a solubilized mitochondrial extract of the COS cells showed substantial activity not only for 25-hydroxylation of 1-hydroxy vitamin D but also for 26-hydroxylation of 5-cholestane-3,7,12-triol.

Lund et al. (595) recently reported cloning of both human and mouse cDNA encoding an enzyme with cholesterol 25-hydroxylase activity. Cells overexpressing the 25-hydroxylase produced 25-OH-Chol from Chol and 24,25-diOH-Chol from 24-OH-Chol. Unlike cytochrome P-450 that catalyze many sterol hydroxylations, the 25-hydroxylase contains a diiron cofactor (Fe-O-Fe or Fe-OH-Fe) bound to histidine clusters. Although it remains to be demonstrated that the primary function of this enzyme is 25-hydroxylation, results reported by Lund et al. (595) with transfected cells overexpressing the 25-hydroxylase appeared to be compatible with the suggested role of 25-OH-Chol in the regulation of cholesterol homeostasis. However, 25-OH-Chol levels in blood and tissues are extremely low (see section IV).

Cali and Russell (153) isolated the human 26-hydroxylase cDNA and determined its sequence. The human 26-hydroxylase was predicted to have an amino acid sequence that showed 81% amino acid homology with the 26-hydroxylase of rabbit liver mitochondria. The combination of Chol (31 µM) and 25-OH-Chol (5 µM) had no effect on the level of mRNA for the 26-hydroxylase in simian virus 40-transformed human fibroblasts. The human 26-hydroxylase cDNA and an expressible cDNA for bovine adrenodoxin were introduced into simian COS cells. Incubation of [7-³H]-5-cholestane-3,7,12-triol with these cells led to the formation of the corresponding C-26 acid as the major product and small amounts of the C-26 alcohol (as judged by TLC). Incubation of [7-³H]-5-cholestane-3,7,12,26-tetrol with the cells led to the formation of the corresponding acid (as judged by TLC). Cali et al. (152) localized the gene for the 26-hydroxylase to chromosome 2 in humans and to chromosome 1 in the mouse. Axen et al. (36) presented evidence indicating that the human cyp26 also catalyzes the 1-hydroxylation and 25-hydroxylation of vitamin D₃. They reported that recombinant expressed human cyp26 in Escherichia coli catalyzed not only the 26-hydroxylation of 5-cholestane-3,7,12-triol and the oxidation of 5-cholestane-3,7,12,26-tetrol to the corresponding acid but also the 25-hydroxylation of vitamin D₃ and of 1-hydroxyvitamin D₃, and the 26- and 1-hydroxylation of 25-hydroxyvitamin D₃. The same workers also reported that COS-1 cells transfected with human cyp26 cDNA catalyzed not only the 26-hydroxylation of 5-cholestane-3,7,12-triol but also (to a lower extent) the 1-hydroxylation and 26-hydroxylation of 25-hydroxyvitamin D₃.

Pikuleva et al. (803) also reported the expression of human cyp26 in E. coli. The purified recombinant enzyme catalyzed the 26-hydroxylation of Chol and, with a much higher efficiency, 5-cholestane-3,7,12-triol. The enzyme was also found to catalyze the conversion of 25-hydroxyvitamin D₃ to material with the HPLC behavior of its 1-hydroxy derivative. The catalytic activity for this reaction was found to be very considerably less than that for the 26-hydroxylation of Chol or of 5-cholestane-3,7,12-triol. It is of interest that studies with recombinant liver cyp26 expressed in E. coli (36, 803, 804) have not noted the formation of either 24- or 25-hydroxylated products after incubation with Chol or 5-cholestane-3,7,12-triol (as seen in incubations with liver mitochondria). No studies have been presented to establish the stereochemistry at C-25 in the 26-hydroxysterol formed with the recombinant enzyme.

P-450 cyp26 from liver mitochondria has been purified to homogeneity in the cases of rabbit (1194), rat (729), and pig (61). The enzyme from pig liver catalyzed the 26-hydroxylation of 5-cholestane-3,7-diol, Chol, and 25-hydroxyvitamin D₃. Structural assignments for the products were based on HPLC. The rat cyp26 has been expressed in yeast (887), and the human cyp26 has been expressed in E. coli (36, 803, 804). The enzyme expressed in yeast catalyzed the 26-hydroxylation of 5-cholestane-3,7,12-triol and the 25-hydroxylation of 1-hydroxyvitamin D₃ (887). The products were characterized by HPLC.

Reiss et al. (842) reported the formation of 26-OH-Chol by bovine aortic endothelial cells. 26-OH-Chol and the corresponding C₂₇ carboxylic acid were identified on the basis of GC-MS studies of metabolites recovered in the media. The levels of 26-OH-Chol and the carboxylic acid in the culture medium were markedly increased by incubation in the presence of added Chol (20 µM). Incubation of the cells with 5-cholestane-3,7,12-triol led to decreased levels of 26-OH-Chol and the C₂₇ carboxylic acid in the medium. Under similar cell culture conditions, the formation of 26-OH-Chol (along with the corresponding C₂₇ and C₂₄ carboxylic acids) was also detected in media from incubations of Hep G2 cells. 26-OH-Chol was not detected in the media after incubation of Chinese hamster ovary (CHO) cells. Björkhem et al. (84) reported a time-dependent increase in the levels of 26-OH-Chol and of 3-hydroxycholest-5-en-26-oic acid in the medium after incubation of human alveolar macrophages for different periods of time in MEM supplemented with 10% heat-inactivated fetal calf serum (FCS). The levels of the 26-OH-Chol and the C₂₇ acid in the cells were reported to be "in general less than 20% and 5%, respectively, of the content of those compounds in the medium." Cyclosporin suppressed the levels of 26-OH-Chol and the C₂₇ carboxylic acid in the medium. The effect on the levels of the cholestenoic acid was more marked than that of the levels of the 26-OH-Chol. With the use of trideuterated Chol (apparently labeled at C-6, C-7, and C-7; although this reviewer could not locate a description of the synthesis of this d₃-Chol), the formation of d₃-26-OH-Chol and d₃-3-hydroxycholest-5-en-26-oic acid was also reported. The presence of the 26-hydroxylase was also shown by Western blot experiments. Björkhem et al. (84) reported that cultured human endothelial cells also formed 26-OH-Chol and 3-hydroxycholest-5-en-26-oic acid, but in amounts very considerably less than they observed with pulmonary macrophages. It was proposed that "conversion of cholesterol into 27-hydroxycholesterol and 3-hydroxy-5-cholestenoic acid represents a general defense mechanism for macrophages and possibly for other peripheral cells exposed to cholesterol. Absence of this defense mechanism may contribute to the premature atherosclerosis known to occur in patients with sterol 27-hydroxylase deficiency (cerebrotendinous xanthomatosis)." Lund et al. (590) extended these studies and reported that, with human alveolar macrophages preloaded with [¹⁴C]Chol, incubation of the cells with media containing FCS resulted in a significant recovery of ¹⁴C in 26-oxygenated products (26-OH-Chol plus 3-hydroxycholest-5-en-26-oic acid) in the culture medium. However, the recovery of ¹⁴C in 26-oxygenated products was only ~10% that of [¹⁴C]Chol in the medium. In other experiments, the macrophages from different individuals were incubated with media containing 10% FCS. With cells from eight subjects, most (~76%) of the Chol was in the free state. Of the 26-oxygenated metabolites, most were found in the medium (82 ± 13% for 26-OH-Chol and 99 ± 1% for 3-hydroxycholest-5-en-26-oic acid). It was stated that all of the 26-oxygenated products "recovered from the cell medium were always unesterified." In another set of experiments, macrophages from four patients were incubated with media containing FCS and cyclosporin (20 µM) for 24 h. Cyclosporin decreased the amounts of 26-OH-Chol plus 3-hydroxycholest-3-en-26-oic acid in the medium from 6.1 ± 1.6 to 0.4 ± 0.1 fmol·cell¹·24 h¹. Under these conditions, cyclosporin increased the Chol levels in the cell from 10.5 ± 1.5 to 17.8 ± 3.2 fmol/cell. The amount of esterified Chol in the cells was low (<8% of total). Lund et al. (590) also presented evidence (based on blood samplings of hepatic vein, portal vein, and a peripheral artery) that indicated a significant arterial-hepatic venous difference for total 26-oxygenated metabolites and suggested uptake by liver of the 26-oxygenated compounds. The authors suggested "a protective role of sterol 27-hydroxylase in the development of atherosclerosis" on the basis of the above findings and the known predisposition of subjects with cerebrotendinous xanthomatosis (CTX) to develop premature atherosclerosis.

Babiker et al. (40) extended this research to a study of the capacity of various cells to secrete 26-oxygenated products, i.e., 26-OH-Chol and cholest-5-en-26-oic acid, into the culture medium as measured by GC-MS analyses. Human macrophages were reported to show a higher capacity to secrete 26-oxygenated products than human endothelial cells. Human lung alveolar macrophages, human monocyte-derived macrophages, and human endothelial cells (n = 3 in each case) showed mean values of 1400, 300, and 38 ng·10⁶ cells¹·24 h¹, respectively. One culture of human fibroblasts showed 20 ng·10⁶ cells¹·24 h¹. As expected, cells (monocyte-derived macrophages or fibroblasts) from patients with CTX showed little or no secretion of 26-oxygenated products (<2 ng·10⁶ cells¹·24 h¹). In the case of the lung macrophages, the great majority of the 26-oxygenated products present in the culture medium (and, to a lesser extent, in the cells) was found as the 26-carboxylic acid. Addition of FCS, cyclodextrin, cyclodextrin plus Chol, albumin, LDL, high-density lipoprotein (HDL), or apolipoprotein (apo) A-I increased the secretion of 26-oxygenated products from the cells. Incubation of the lung macrophages with 25-OH-Chol (12.4 µM) decreased the secretion of 26-oxygenated products. Westman et al. (1193) conducted additional investigations of the role of 26-OH-Chol (and its corresponding acid) in the net efflux of Chol from monocyte-derived macrophages. Cells loaded with Chol (from acetylated LDL) showed significant levels of 26-OH-Chol, and the efflux of the 26-hydroxysterol was proportional to its intracellular level. The export of Chol and 26-OH-Chol (and the corresponding acid) was markedly affected by the presence and the nature of acceptor species in the culture medium. Incubation with reconstituted HDL or native HDL resulted in a decreased efflux of 26-OH-Chol and cholestenoic acid from the cells and increased the efflux of free Chol into the medium. Because HDL is commonly considered to be an important physiological acceptor of sterols after their efflux from cells, these experiments raise a question as to the importance of the conversion of Chol to 26-OH-Chol and the efflux of 26-OH-Chol as an important protective defense mechanism in the net removal of Chol from cells.

The immunosuppressant cyclosporin A has been reported (823) to show high potency in the inhibition of the 26-hydroxylation of Chol in rat liver mitochondria, with an IC₅₀ value of 4 µM. In contrast, direct addition of cyclosporin A (50 µM) had no effect on cyp7 activity in vitro (823). Additional studies with rat hepatocytes indicated that cyclosporin A caused a concentration-dependent inhibition of the incorporation of [¹⁴C]Chol into total bile acids, with an IC₅₀ value of ~10 µM. The formation of labeled -muricholic acid and of mono- and dihydroxylated bile acids was inhibited considerably more than the synthesis of cholic acid and other polar acids considered to be derived largely from cholic acid. Cyclosporin A (10 µM) also decreased the levels of total bile acids in rat hepatocytes plus media. The decrease in the levels of total bile acids was found to largely reflect decreases in the amounts of chenodeoxycholic acid and -muricholic acid since the levels of cholic acid did not change in the treated cells. The changes in bile acid formation and the major effect on chenodeoxycholic acid and -muricholic acid formation were interpreted as being in accord with postulations of an alternative pathway for the biosynthesis of bile acids apart from the pathway in which 7-hydroxylation of Chol is the initial reaction. It is important to note that cyclosporin A had other effects relative to Chol metabolism in the rat hepatocyes. Whereas cyclosporin A (10 µM) had no effect on the levels of ATP in rat hepatocytes, the antibiotic caused a 14% decrease in the concentration of cellular ATP at 20 µM cyclosporin A. The antibiotic (10 µM) also caused a 46% increase in the amount of cell-associated ¹⁴C after incubation of the rat hepatocytes with [¹⁴C]Chol and a 30% decrease in the incorporation of [2-¹⁴C]acetate into Chol (although methodology on the latter point was not presented).

Dahlbäck-Sjöberg et al. (231) reported that cyclosporin A inhibited the formation of 26-OH-Chol from Chol with either rat liver mitochondria or with a purified 26-hydroxylase from rabbit liver mitochondria. The antibiotic caused a 50% inhibition of 26-OH-Chol formation at ~4 µM. Whereas cyclosporin A was highly active in the inhibition of 26-hydroxylation of Chol and 7-OH-Chol with the rat liver mitochondria, it had little or no effect on the 26-hydroxylation of 5-cholestane-3,7-diol and 5-cholestane-3,7,12-triol. With the purified 26-hydroxylase, the antibiotic also had no significant effect on the 26-hydroxylation of 5-cholestane-3,7,12-triol. Kinetic studies with the purified cytochrome P-450 showed that the inhibition of 26-hydroxylation of Chol by cyclosporin A was noncompetitive in nature. Winegar et al. (1202) reported that cyclosporin inhibited 26-OH-Chol formation by Hep G2 cells (IC₅₀ = 1 µM) and by mitochondria from Hep G2 cells (inhibition constant = 0.25 µM). Identification of the product was based on the incorporation of [¹⁴C]Chol (complexed with -cyclodextrin) into 26-OH-Chol as judged by HPLC. The chromatograms presented did not include information of the mobilities of other oxygenated sterols such as 25-OH-Chol or 24-OH-Chol (which are expected to be formed in mitochondrial incubations) or other oxysterols that might arise from autoxidation of [¹⁴C]Chol during incubations and/or sample processing. The cyclosporin used was apparently a commercial preparation for intravenous administration (containing substantial amounts of a polyoxyethylated castor oil) that was then added in dimethyl sulfoxide. The authors reported that, with freshly isolated mitochondria from Hep G2 cells, the inhibition caused by cyclosporin of 26-OH-Chol formation was competitive. It should be noted that cyclosporin itself undergoes metabolism by cytochrome P-450 systems similar to those involved in the initial steps of the side-chain oxidation of sterols, i.e., hydroxylation and oxidation to aldehydes and carboxylic acids (205). Winegar et al. (1202) also reported that cyclosporin had other actions upon incubation with Hep G2 cells. Cyclosporin, at 2.5 and 10 µM, inhibited the incorporation of [¹⁴C]Chol into Chol esters of cells (19 and 32%, respectively); this was associated with increased levels of free [¹⁴C]Chol in the cells (+26% at 2.5 µM and +75% at 10 µM). Cyclosporin, at 0.25 and 1.0 µM, had no significant effect on the esterification of Chol. Winegar et al. (1202) also reported that cyclosporin, at 10 µM (but not at 1 µM), caused an inhibition of LDL uptake (as measured by cell-associated ¹²⁵I-LDL). In these studies, possible effects of the castor oil derivative in the commercial sample of cyclosporin were not evaluated. Holmberg-Betsholtz and Wikvall (394) reported that purified rabbit liver cyp26 catalyzed the conversion of Chol to 26-OH-Chol, but the activity of the enzyme with this substrate was considerably less than that with 5-cholestane-3,7-diol or 5-cholestane-3,7,12-triol. Incubation in the presence of carbon monoxide (CO:O₂, 98:2; vs. N₂:O₂, 98:2) inhibited the 26-hydroxylation of 5-cholestane-3,7,12-triol. Ketoconazole (an inhibitor of cytochrome P-450), but not disulfiram (an inhibitor of aldehyde dehydrogenase), inhibited the 26-hydroxylation of 5-cholestane-3,7,12-triol with the purified rabbit liver cyp26.

Cyclosporin administration has been reported to increase plasma Chol levels (9). Whether or not this effect is due to the inhibitory action of cyclosporin on the conversion of Chol to 26-OH-Chol is not known. 26-OH-Chol is a potent downregulator of HMG-CoA reductase and an inhibitor of sterol synthesis. In addition, as noted previously, the conversion of Chol to 26-OH-Chol has also been suggested to be important in providing a mechanism for the elimination of excess Chol from cells (84, 590).

Preincubation of mitochondria with several peripheral benzodiazepine receptor ligands or the induction of hepatic porphyria resulted in decreases in the conversion of labeled Chol to 26-OH-Chol (as judged by radio-TLC analysis) by rat liver mitochondria (1128). The effects of these treatments on the formation of 26-OH-Chol were ascribed to modulation of the intracellular transport and availability of the Chol substrate.

The presence of 26-hydroxylase has been demonstrated in frozen and thawed preparations of human skin fibroblasts (992). The following sterols were reported to be substrates for the 26-hydroxylase: 5-cholestane-3,7,12-triol, 5-cholestane-3,7-diol, 7-OH-Chol, and 7-hydroxycholest-4-en-3-one. Chol was not tested as a substrate.

Axelson and Larsson (32) reported the formation of 26-OH-Chol after incubation of FCS with normal human fibroblasts. Identification of the 26-OH-Chol was based on GC-MS of its TMS derivative. All GC-MS experiments involved the addition of an internal standard of a sample of 26-OH-[²H]Chol containing predominantly 7 atoms of deuterium. Almost all of the 26-OH-Chol reported to be formed was found in the medium. Increases in the amounts of 26-OH-Chol found in the medium at 24 h were ~3.5 times that found in cells incubated for only 15 min with FCS. Under the conditions studied, the levels of 25-OH-Chol and of 24-OH-Chol in the medium (or in cells) at 24 h did not appear to be different from those found after 15 min of incubation with FCS. Substantial levels of 7-keto-Chol were found in medium (and in cells) after either 15 min or 24 h of incubation of the cells with FCS. For example, the levels of 7-keto-Chol in medium at 15 and 24 h were ~12 and ~3 times, respectively, that of the levels of 26-OH-Chol in the same experiment. Significant levels of 7-OH-Chol and 7-OH-Chol were also detected in medium (and in cells) after incubation of the cells with FCS for 15 min or 24 h. Cyclosporin A appeared to abolish the increase in 26-OH-Chol found in the medium. Also, little or no increase in 26-OH-Chol in medium was observed upon incubation of cells from a homozygous FH subject. No analyses of the oxysterol levels in the FCS used were presented. Experiments with [³H]Chol and/or [³H]Chol oleate incorporated into FCS, and analysis by HPLC also showed formation of 26-OH-[³H]Chol which was almost exclusively in medium. The formation of 26-OH-[³H]Chol under these conditions appeared to be substantially suppressed by cyclosporin A. It should be noted that only a small fraction of the labeled Chol was converted to 26-OH-Chol (~0.04% of the internalized LDL Chol). A study of the time course of the effects of the addition of FCS to fibroblasts on the levels of newly formed 26-OH-Chol (in the medium) and the levels of HMG-CoA reductase suggested that the changes might be related. However, the major decreases in reductase activity occurred before any substantial increase in 26-OH-Chol in the medium. Axelson and Larsson (33) described other experiments indicating the formation of 26-OH-Chol and, in lesser amounts, 25-OH-Chol from Chol of FCS (with ~70% of the Chol in LDL) in normal human fibroblasts (based on GC-MS analyses of compounds in the culture media). Also formed were significant amounts of cholest-5-ene-3,7,26-triol, 7,26-dihydroxycholest-4-en-3-one, 3-hydroxy-cholest-5-en-26-oic acid, 3,7-dihydroxycholest-5-en-26-oic acid, 3-oxo-7-hydroxycholest-4-en-26-oic acid, 3,7-dihydroxycholest-5-en-26-oic acid, and 3-hydroxy-7-oxo-cholest-5-en-26-oic acid. The major 26-oxygenated sterols detected were 26-OH-Chol, 7,26-dihydroxycholest-4-en-3-one, 7,26-dihydroxycholest-5-en-7-one, and 3-oxo-7-hydroxycholest-4-en-26-oic acid. Quite different results were observed with a virus-transformed line of human fibroblasts. In this case, very little formation of the 26-oxygenated sterol or its metabolites was observed. In the same experiments, substantial amounts (in order of abundance) of 7-keto-Chol, 7-OH-Chol, and 7-OH-Chol were observed in the culture media from 15-min and 48-h incubations of both the normal fibroblasts and the virus-transformed fibroblasts with the FCS. The authors noted that "most of these sterols were probably formed by autoxidation of cholesterol during the purification of the samples, since their amounts did not differ significantly from those of the controls (15-min incubations)." This may be the case. In addition, the presence of these oxysterols in the FCS used in these experiments was not excluded since analyses of the FCS were not made. In the normal human fibroblasts cyclosporin A inhibited the formation of 26-OH-Chol, 7,26-dihydroxycholest-4-en-3-one, 7-hydroxycholest-4-en-26-oic acid (as judged by HPLC) from labeled Chol or labeled Chol oleate (preincubated with FCS before cellular incubations). It is important to note that the results of the experiments of Axelson and Larsson (32, 33) were interpreted as indicating the formation of 26-OH-Chol from Chol (or its esters) present in LDL. Whereas this is probably the case, the actual experiments did not involve incubation of the cells with preparations of isolated LDL; they involved incubations with FCS.

Reiss et al. (843) reported on the expression of sterol 26-hydroxylase in human endothelial cells from pulmonary artery and aorta as judged by measurement of levels of mRNA. mRNA for the 26-hydroxylase was also detected in Hep G2 cells but not in CHO-K1 cells. 26-OH-Chol and 3-hydroxycholest-5-en-26-oic acid were observed in the culture media in the cases of endothelial cells (from both pulmonary artery and aorta) incubated for 72 h in media containing delipidated FCS. Increased levels of the two compounds in the culture medium were observed after incubations of the endothelial cells in the presence of added Chol. The formation of 26-OH-Chol and 3-hydroxycholest-5-en-26-oic acid was also observed after incubation of Hep G2 cells for 72 h with exogenous Chol in media containing delipidated FCS. The level of 26-OH-Chol in the medium of the Hep G2 cells was comparable to that observed for endothelial cells from aorta (grown in the same medium containing added Chol). Under the conditions studied, the level of 3-hydroxycholest-5-en-26-oic acid in the medium for the Hep G2 cells was ~10 times higher than that observed for the aortic endothelial cells. The authors proposed that "27-hydroxylase activity in arterial endothelium provides a local mechanism of defense against Chol accumulation within the arterial wall by serving as a pathway for elimination of intracellular Chol by conversion to more polar metabolites," a suggestion bearing remarkable similarity to the suggestion made by Björkhem et al. (84) for results obtained from human pulmonary alveolar macrophage cells. Reiss et al. (843) did not detect formation of 26-OH-Chol in the medium after incubation of CHO cells for 72 h in medium containing delipidated FCS and added Chol. Whereas the 26-OH-Chol was not detected, low levels of 3-hydroxycholest-5-en-26-oic acid were reported.

Taurocholic acid (10, 30, and 50 µM) caused a significant reduction in the level of cyp26 activity in rat hepatocytes (1137). The extents of inhibition were 31 and 81% at concentrations of 10 and 50 µM taurocholate, respectively. Taurocholate (50 µM) also lowered the levels of cyp26 mRNA, and there was an even more striking decrease in the level of mRNA for cyp7a. No effect of taurocholate on the level of mRNA for lithocholic acid 6-hydroxylase was observed. The lowering of mRNA for the 26-hydroxylase by taurocholate was dose dependent with significant reduction at 5-50 µM. Maximal lowering of cyp26 mRNA was observed at 30 and 50 µM. The concentrations of the taurocholate used were stated to be in the physiological range reported in portal vein blood of rats. Taurocholate, cholic acid, and deoxycholic acid, each at 50 µM, had essentially identical effects in lowering the levels of cyp26 mRNA in cultured rat hepatocytes. Chenodeoxycholic acid (50 µM) was less potent, and -muricholic acid (50 µM) had no significant effect. Taurocholate (50 µM) suppressed transcriptional activity for both cyp26 and for cyp7a. In another paper, the same group (1139) extended these studies. At 50 µM, either deoxycholic acid or cholic acid resulted in marked lowering of cyp26 activity, mRNA levels, and transcriptional activity in rat hepatocytes. Similar findings were made with cyp7a. In contrast to these effects of deoxycholate and cholate, little or no effects were observed with -muricholic acid and ursocholic acid, two hydrophilic bile acids. In contrast to these studies with cultured cells, Shefer et al. (966) reported that feeding deoxycholic acid to rats had no effect on the levels of hepatic cyp26 activity or on the levels of mRNA for this enzyme. In contrast, marked lowering of the levels of cyp7a activity was observed that was associated with a striking increase in the levels of mRNA for this enzyme. Xu et al. (1208) reported that the levels of hepatic mitochondrial cyp26 activity of New Zealand White (NZW) rabbits increased by +66% upon Chol feeding (2% Chol in a chow diet) for 10 days. Total biliary drainage for 7 days had no effect on the levels of the cyp26 activity in chow-fed or in the Chol-fed animals. The assay used was based on the incorporation of [4-¹⁴C]Chol into material with the TLC mobility of 26-OH-Chol and recrystallization to constant specific activity (data not presented). Xu et al. (1209) recently reported increased cyp26 activity in NZW and heterozygous (but not homozygous) WHHL rabbits on Chol feeding (3 g/day for 10 days).

Post et al. (815) observed that cafestol, a diterpene responsible for the hypercholesterolemic effect of boiled coffee, caused a dose-dependent decrease in cyp26 activity, bile acid synthesis, and cyp7 activity in cultured rat hepatocytes. The decrease in 26-hydroxylase activity was associated with decreases in its mRNA levels and in cyp26 gene transcriptional activity. Clear effects on each of the above were observed at 32 µM cafestol. Modest decreases (~20%) in mRNA levels for LDL receptor and HMG-CoA reductase were observed at 63 µM cafestol.

Twisk et al. (1138) reported that insulin, at physiological concentrations, inhibited the formation of bile acids and lowered cyp26 activity. The decrease in enzyme activity caused by insulin was associated with a decrease in the level of mRNA for the enzyme and decreased transcriptional activity. Very similar results were obtained with respect to the effects of insulin on cyp7a.

Sterol 26-hydroxylase activity is preferentially localized in the pericentral hepatocytes in rat liver (1140). The enzyme activity, steady-state mRNA level, and gene transcription for cyp26 were reported to be 2.9, 2.5, and 1.7 times higher, respectively, in pericentral hepatocytes than in periportal hepatocytes. A similar preferential localization in pericentral hepatocytes has also been reported for cyp7 (see sect. IIIB).

Hasan and Kushwaha (376) reported the formation of 26-OH-Chol in homogenates of baboon liver in the presence of added NADPH-generating system. Identification of the 26-OH-Chol was limited to its behavior on a reverse-phase HPLC system for which only 26-OH-Chol, 7-keto-Chol, and Chol were used as authentic standards. The nature of the homogenate preparations was not specified except to note that they were prepared from frozen (70°C) samples of liver and that "homogenates were prepared in phosphate-buffered saline by polytron homogenizer on ice." The methodology for quantitation of the 26-OH-Chol involved saponification of the incubation mixture followed by reverse-phase HPLC. The base-labile 7-keto-Chol was added to the strongly alkaline saponified incubation mixture before extraction of the nonsaponifiable lipids (NSL). The levels of cyp26 activity were measured as the difference between results obtained at 4 and 37°C. The substrate involved in these assays was apparently Chol present in the homogenate preparation, since no addition of exogenous sterol was mentioned. The magnitude of "background" cyp26 activity at 4°C was not presented. Using this methodology, Hasan and Kushwaha (376) reported that the mean value of cyp26 activity of baboon liver homogenates was higher in samples from "low responders" to a high-fat, high-Chol diet [318 ± 95 (SE) µg 26-OH-Chol·g liver¹·h¹; n = 3] than in those from "high responders" [67 ± 13 (SE) µg 26-OH-Chol·g liver¹·h¹; n = 3]. The animals were fed the high-fat, high-Chol diet for an unspecified period of time. Kushwaha et al. (526) reported on the levels of mRNA for the cyp26 in livers of baboons on a chow diet and at various times after the administration of a high-fat (40% of calories), high-Chol (1.7 mg/kcal) diet. Three groups of animals were employed that differed in their levels of serum Chol upon administration of the high-Chol diet. The levels of cyp26 mRNA in the livers of low responders on the chow diet were higher than those observed in the medium responders and high responders. Upon changing from the chow diet to the high-Chol, high-fat diet, little or no change in 26-hydroxylase mRNA was observed in the high responders. In contrast, significant increases were observed in the low responders and medium responders. The cyp26 mRNA levels in the medium and low responders on the high-fat, high-Chol diet were higher than those of the high responders at each of the time points studied. Except for the value at 3 wk after initiation of the high-fat, high-Chol diet, the levels of 26-hydroxylase mRNA in the low responders were higher than those of the medium responders. In the same study the low responders and the high responders did not differ with respect to the levels of mRNA in liver for cyp7a on the chow diet or on the high-fat, high-Chol diet (weeks 3, 10, and 78). Furthermore, no differences were observed between the levels of mRNA for cyp7a upon changing from a chow diet to the high-Chol diet (in either the low responders or the high responders). Chen et al. (190) reported increases in the levels of cyp26 activity and protein in low responder baboons on a high-Chol, high-fat diet after 6 and 10 wk (relative to values obtained at 3 wk). In the high responders, no significant increases were observed at 6 and 10 wk. Unfortunately, no data were provided on animals before the administration of the high-Chol, high-fat diet. Another study (527) from the same laboratory concerned studies in ovariectomized baboons on the high-Chol, high-fat diet. Hepatic and adrenal levels of mRNA for the cyp26 were higher in animals treated with estrogen (estradiol cypionate) or with the combination of estrogen plus progesterone. Wang et al. (1181) reported that hepatic mitochondrial cyp26 activity in piglets was decreased by fasting and increased by feeding (nursing).

Sugawara et al. (1066) observed that expression of steroidogenic acute regulatory protein in COS-1 cells cotransfected with cyp26 and adrenodoxin caused a marked (6-fold) increase in the formation of 3-hydroxycholest-5-en-26-oic acid. The steroidogenic acute protein stimulates steroid formation in adrenal and gonadal cells and appears to be involved in the transport of Chol within mitochondria. The results of Sugawara et al. (1066) indicate that this protein can stimulate not only steroid hormone formation from Chol, but it can also stimulate the conversion of Chol to 26-OH-Chol and the cholestenoic acid. Other recent investigations of the regulation of the 26-hydroxylase of rat liver have indicated sequence complementarity between the 5'-terminal regions of the mRNA for the rat liver mitochondria 26-hydroxylase and serine protease inhibitors (960, 961), the levels of the latter previously shown (1216) to be regulated by growth hormone. The sequence overlap was noted to "probably represent the first observation of 5'-end overlap between two functional mRNA" (960). The mRNA levels of the 26-hydroxylase in liver were shown to be regulated by growth hormone (961). Further studies indicated that the mRNA for the serine protease inhibitors can regulate the expression of the cyp26 gene (960).

Bile duct ligation in male Sprague-Dawley rats has been reported (364) to result in increased in vitro 26-hydroxylation of Chol by liver mitochondria.

The importance of the 26-hydroxylase is clearly demonstrated in the case of CTX, an autosomal recessive sterol storage disease characterized by tendon xanthomas, neurological dysfunction with dementia and cerebellar ataxia, and premature atherosclerosis and cataracts (85). Homozygous subjects show very low levels or the absence of 26-hydroxylase activity in fibroblasts obtained by skin biopsy (152, 489, 992). A number of different mutations in the cyp26 gene have been reported (6, 152, 191-193, 323, 489, 553, 694, 1167, 1168). Very recently, Rosen et al. (862) described studies of mice with a disrupted sterol 26-hydroxylase gene. The level of bile acids in feces and bile was very much lower in a cyp26 / mouse than in a control (cyp26 +/+) mouse. Furthermore, the conversion of [7-³H]7-OH-Chol to acidic material in feces was much lower in a cyp26 / mouse than in a control (cyp26 +/+) mouse after its intraperitoneal injection. The mean level of 7-OH-Chol in serum was reported to be elevated in the cyp26 / mice (n = 6) when compared with that for control (cyp26 +/+ mice; n = 5), with mean values of 5.0 ± 2.5 and 1.2 ± 0.5 (SE) µM, respectively. However, from the data presented, these mean levels did not differ significantly (P > 0.05). The mean level of 26-OH-Chol in serum from the cyp26 / mice was reported as <0.0025 µM as compared with 0.20 ± 0.03 µM for the control mice. The levels of 7-OH-Chol in liver, brain, and kidney were reported to be higher in cyp26 / mice than in control mice, although it was not specified as to whether the results presented corresponded to analyses of pool samples or from individual animals from each group. The mean level of serum lathosterol, taken as an indirect measure of hepatic HMG-CoA reductase activity, was reported to be significantly elevated in the knockout mice, and the hepatic level of HMG-CoA reductase mRNA was stated to be two- to threefold higher in the knockout mice. Interestingly, indications of neurological or vascular abnormalities or marked elevation of tissue Chol, as seen in human CTX, were not detected.

The results of an important early study in 1958 by Bergström et al. (62) demonstrated that the 7-hydroxylation involved in the overall conversion of Chol to cholic acid occurs with stereospecific loss of the 7-hydrogen. In 1965, Mendelsohn et al. (642) reported the conversion of [26-¹⁴C]Chol to labeled 7-OH-Chol and 7-OH-Chol upon incubation with rat liver preparations. Identification of the labeled products rested largely on the results of cocrystallization studies. The authors ascribed the formation of both of the 7-hydroxysterols to enzymatic action and not to autoxidation of Chol. However, it should be noted that the reported incorporations of the labeled Chol into 7-OH-Chol (i.e., ~1.1%) and 7-OH-Chol (i.e., ~0.09%) were quite low and were comparable to that of the incorporation into 7-OH-Chol with one heat-inactivated control incubation. The possibility that the 7-oxygenated derivatives of Chol arose from autoxidation of Chol under the incubation conditions employed is a very important matter in view of prior work of Danielsson (233). In another early study, Björkhem et al. (87) reported, upon incubation of labeled Chol with the 20,000-g supernatant fraction of a rat liver homogenate, the formation of the following oxygenated sterols: 7-OH-Chol, 7-keto-Chol, 7-hydroxycholest-4-en-3-one, 7,12-dihydroxycholest-4-en-3-one, and 5-cholestane-3,7,12-triol. The labeled products were characterized on the basis of TLC and cocrystallization experiments. Although the incubations were relatively short, no rigorous controls to assess the possible artifactual formation of 7-OH-Chol and 7-keto-Chol from autoxidation of Chol were employed. The formation of 7-OH-Chol, 7-OH-Chol, and 7-keto-Chol from Chol has also been reported (454) with rat liver microsomes in the presence of NADPH under conditions permitting peroxidation of lipids (i.e., in the absence of EDTA).

7-OH-Chol, 7-OH-Chol, and 7-keto-Chol, as well as the 7-hydroperoxides, are products of the autoxidation of Chol. An abundance of evidence has demonstrated that 7-OH-Chol is also an enzymatic product. Whether or not 7-OH-Chol is also the product of direct enzymatic action is less clear. Although Mendelsohn et al. (642) claimed the indication of the presence of Chol 7-hydroxylase activity in rat liver preparations, the evidence presented on this point was very limited and inconclusive. Shoda et al. (976) reported that rat liver mitochondria have the capacity to epimerize certain 7-hydroxysterols or their cholest-5-en-26-oic acid analog to give the corresponding 7-hydroxy compounds. The epimerization was proposed to occur via 7-keto intermediates. The action of rat liver mitochondria on 7-OH-Chol itself was apparently not studied. The enzymatic nature of the formation of the 7-hydroxy compounds was indicated by the dependence of the reaction on the presence of added isocitrate and the amount of mitochondrial protein. Breuer and Björkhem (123) reported substantial enrichment of ¹⁸O in both oxygens of 7-OH-Chol of liver and plasma after exposure of rats to ¹⁸O₂. In the same experiments, very substantial enrichment of 5-cholest-7-en-3-ol with ¹⁸O was observed; however, the ¹⁸O content of Chol was minimal. The results (from 2 rats) were interpreted as demonstrating the formation of 7-OH-Chol from Chol in vivo and suggested the possibility that the 3,7-dihydroxy-⁵-sterol "is predominantly formed from newly synthesized cholesterol, or a cholesterol precursor, by enzymatic reactions in vivo."

Song et al. (1015) reported the enzymatic conversion of 7-OH-Chol to 7-keto-Chol with hamster liver microsomes in an NADP-dependent reaction. This enzyme activity was also present in liver microsomes of human and bovine origin but was absent in liver microsomes of rat, mouse, and rabbit. 7-OH-Chol was not tested as a substrate. Thus 7-keto-Chol can arise not only from decomposition of the 7- and 7-hydroperoxides of Chol but also by enzymatic dehydrogenation of 7-OH-Chol (at least with hamster, human, and bovine liver microsomes). Song et al. (1014) purified this enzyme from hamster liver microsomes. The purified enzyme catalyzed the C-7 dehydrogenation of both 7-OH-Chol and 7-OH-Chol to form 7-keto-Chol. It also efficiently catalyzed the C-11 dehydrogenation of hydrocortisone and corticosterone (11-hydroxysteroids). The purified enzyme was also reported to catalyze the C-3 dehydrogenation of Chol. The hamster 7-OH-Chol dehydrogenase showed high sequence similarity to human 11-hydroxysteroid dehydrogenase. Although low or absent in rat liver microsomes, immunochemical studies indicated the presence of 7-OH-Chol dehydrogenase in human liver microsomes. This was the first description of an enzyme with 7-hydroxysterol dehydrogenase activity and provides an origin of 7-keto-Chol other than by autoxidation of Chol or peroxidative metabolism.

The formation of 7-oxygenated sterols from Chol has also been reported upon incubation of Chol and ethyl linoleate in the presence of horseradish peroxidase. Teng and Smith (1106) reported that the 7- and 7-hydroperoxides were the initial and chief products. Formation of 7-oxygenated sterols from Chol has also been reported upon incubation of Chol with soybean lipoxygenase in the presence of linoleic acid or ethyl linoleate (454, 594, 1106). Teng and Smith (1106) reported the formation of the 7- and 7-hydroperoxides (1:3 to 2:3 ratio of 7- to 7-products) of Chol upon incubation of [³H]Chol and ethyl linoleate with soybean lipoxygenase. The 7- and 7-hydroperoxides are thermolabile, and each undergoes thermal decomposition to 7-keto-Chol and to 7- and 7-OH-Chol (and to a number of other lesser products) (558). Johansson (454) reported the formation of 7-OH-Chol, 7-OH-Chol, and 7-keto-Chol (as judged by radio-TLC) after incubation of labeled Chol with soybean lipoxygenase and linoleic acid. The relative amounts of the three sterols formed were ~1.0:1.1:2.9, respectively. Lund et al. (594) reported the formation of 7-OH-Chol, 7-OH-Chol, and 7-keto-Chol upon incubation of Chol with lipoxygenase in the presence of linoleic acid. The 7- and 7-hydroxysterols were reported to be formed in approximately the same amounts. The 5,6- and 5,6-epoxides of Chol and 5,6-diOH-Chol were also formed under these conditions. Identification of products was based on behavior on HPLC and GC-MS studies (spectral data not presented). The formation of 7-OH-Chol, 7-OH-Chol, 7-keto-Chol, and the 5,6- and 5,6-epoxides of Chol has also been reported upon incubation of Chol with 13-hydroperoxyoctadeca-9,11-dienoic acid (594).

Chol 7-hydroxylase (cyp7a) from different species has been purified (197, 449, 724), cloned (198, 210, 221, 449, 564, 718, 1131), and sequenced (198, 210, 221, 449, 564, 718, 719, 1131, 1178), and the cyp7a from rats and humans have been expressed in E. coli and purified (477, 565). A number of discrepancies in reported nucleotide sequences for the human cyp7a gene have been noted (1178). Several reviews of factors controlling cyp7a activity have been presented (93, 876, 1172). Chiang and Stroup (199) reported the identification of a putative bile acid-responsive element in the cyp7a gene promoter of rat liver. The cyp7a activity is localized primarily in the pericentral hepatocytes of rats (67, 1140, 1143). Enzyme activity, steady-state mRNA, and gene transcription for the 7-hydroxylase were 7.9, 9.9, and 4.4 times higher in the pericentral hepatocytes than in periportal hepatocytes (1140). Very recently, Massimi et al. (622) reported that the localization of cyp7a mRNA in rat liver shows marked changes during development, with a homogeneous distribution at birth followed by changes to the adult pattern of pericentral localization at 28 days. A striking diurnal variation in the levels of enzyme activity, enzyme protein, and mRNA has been presented for the 7-hydroxylase in liver of normal rats (462, 719) and for enzyme activity and mRNA in rabbit liver (462), and it has been suggested that pretranslational regulation is responsible for the circadian rhythm of the 7-hydroxylase (719). The basal (10 A.M.) rat hepatic mRNA expression and gene transcription appears to be localized to only four or five hepatocytes of the liver cell plate located near the hepatic venules (rather than portal venules) (67). At the time of highest mRNA expression and gene transcription of the cyp7a gene (~10 P.M.), about one-half to two-thirds of the hepatocytes containing detectable mRNA for the 7-hydroxylase were located near the hepatic venule. A diurnal variation of the levels of cyp7a (120) and of cyp7a mRNA in liver of cholestyramine-fed rats has also been presented (564). Starvation reduced the level of cyp7a activity of rat liver microsomes in male rats (719). Chol feeding and cholestyramine administration increased the levels of mRNA in liver for the 7-hydroxylase (120, 449, 564, 702, 753, 874). Chol feeding (2% in diet) of rats for 14 days resulted in increases in microsomal cyp7a activity (+288%), mRNA levels in liver (+291%), and liver nuclear transcriptional activity (+220%) for the 7-hydroxylase (449). Feeding of either cholic acid or chenodeoxycholic acid, at a level of 1% in diet, decreased the levels of hepatic mRNA for the 7-hydroxylase. In contrast to the case of the cyp26 (11), cyp7a mRNA was detected only in liver (449) and was not detected in testes, adrenal, kidney, heart, lung, or brain. However, in another study (197), cyp7a activity was reported not only in liver but also in kidney, heart, and lung, albeit at lower levels than in liver.

Chol feeding results in increased levels of hepatic cyp7a activity in rats (82, 88 and references cited therein) and pigs (298). Björkhem et al. (88) reported that Chol feeding (2% in diet) increased cyp7a activity in livers of rats. At a lower level (1% in diet), Chol had no significant effect on enzyme activity. Interestingly, Chol feeding (2% in diet) increased the levels of cyp7a activity in liver in thoracic duct-cannulated rats. It was suggested that the Chol-induced increase in enzyme activity was due to factors "unrelated to the flux of Chol from the intestine to the liver." It was suggested that high levels of dietary Chol resulted in "increased binding of bile acids in the intestine and increased loss of bile acids in feces." The loss of bile acids was proposed to "lead to a reduced suppression of the cholesterol 7-hydroxylase by the bile acids." Osada et al. (735) reported that dietary administration of Chol (0.5%) for 21 days to young or adult male rats was associated with a higher level of cyp7a activity in liver microsomes (relative to animals on a Chol-free diet).

It is important to note that Chol feeding does not always induce increases in the levels of cyp7a and/or cyp7a mRNA in liver (184, 512, 814, 873, 993, 1141). Administration of a high-Chol (1.7 mg/kcal), high-fat (40% of calories) diet to baboons had no effect (at weeks 3, 10, and 78) on the levels of cyp7a mRNA in liver (relative to values on a chow diet) (526). Furthermore, no differences were observed between high and low responders to dietary Chol (while on the chow diet or the high-fat, high-Chol diet) (993). Krause et al. (512) presented results indicating that NZW rabbits fed a chow diet supplemented with Chol (0.5%), peanut oil (3%), and coconut oil (3%) for 8 wk, sufficient to induce marked hypercholesterolemia, did not show an increase in hepatic cyp7a mRNA. In fact, an apparent decrease in the level of mRNA was observed that was of borderline statistical significance. Poorman et al. (814) reported that feeding Chol (0.25% in diet) had no effect in rabbits on the levels of hepatic cyp7a activity or on the levels of mRNA for this enzyme. Rudel et al. (873) reported substantially lower values of hepatic cyp7a activity and mRNA upon feeding of a high-Chol (0.8 mg Chol/kcal) diet relative to control animals receiving a monkey chow diet (low Chol, low fat). Xu et al. (1207) reported that feeding Chol, either 0.2% or 2% in a chow diet for 10 days, decreased the levels of hepatic microsomal cyp7a activity 41 and 53%, respectively. Chol feeding (2%) lowered the levels of mRNA (79%). In animals on a chow diet, the mean level of cyp7a activity and mRNA were lower in Watanabe heritable hyperlipidemic (WHHL) rabbits than in NZW rabbits (1207). Xu et al. (1208) reported that the levels of hepatic microsomal cyp7a activity decreased (68%) in NZW rabbits upon Chol feeding (2% Chol in a chow diet) for 10 days. Total biliary drainage for 7 days resulted in increases in cyp7a activity in both chow-fed control rabbits and the Chol-fed rabbits. The decreased levels of cyp7a activity in the Chol-fed rabbits were attributed to an increased bile acid pool resulting from an associated increase in hepatic cyp26 activity. Xu et al. (1209) recently reported decreased cyp7a activity in NZW and heterozygous WHHL rabbits after Chol feeding (3 g/day for 10 days).

Horton et al. (401) reported marked differences in the level of cyp7a activity in livers of rats and hamsters fed the same Chol-free diet. The levels in rats were ~16-fold higher than those in hamsters, a finding that was associated with a 20-fold higher synthesis of Chol in the rat, as measured by the incorporation of labeled hydrogen into digitonin precipitable sterols (DPS) in liver at 1 h after the intravenous injection of ³H-labeled water. Chol feeding resulted in a decrease in Chol synthesis in both species. However, the induction by Chol feeding of increased levels of cyp7a expression in the rat was not observed in the hamster.

Cheema et al. (184) presented important results that might explain at least a portion of the variable responses of cyp7a gene expression and enzyme activity after Chol feeding. Their studies, conducted in female mice (C57BL/6J), demonstrated that the responses to Chol administration (1% in diet) varied markedly in animals fed diets containing different fats [none or diets with a high level (20% by weight) of fats] containing either polyunsaturated fatty acids (safflower oil), monounsaturated fatty acids (olive oil), or saturated fatty acids (beef tallow). Mice fed Chol (1%) in a chow diet for 3 wk showed a very large increase in cyp7 mRNA in liver. However, administration of Chol (1%) in the three high-fat diets resulted in different responses. Animals on the diet high in fat containing polyunsaturated fatty acids showed significant increases in cyp7a mRNA and enzyme activity (relative to controls on the same diet). In contrast, animals on either the diet with fats high in monounsaturated fatty acids or saturated fatty acids showed significant decreases in cyp7a mRNA and enzyme activity relative to control animals on the same diets. Cheema et al. (184) noted that administration of the high-fat diets alone (without added Chol) resulted in increases in cyp7a mRNA and enzyme activity. Thus the results noted above reflect the findings that the addition of Chol to the various diets resulted in further increases in cyp7a mRNA and enzyme activity with the polyunsaturated fatty acid diet but resulted in a lowering of these values in the animals fed the monounsaturated or saturated fatty acid-rich diets.

Björkhem et al. (96) reported that liver microsomes from human subjects catalyzed the conversion of 26-OH-Chol to cholest-5-ene-3,7,26-triol. Evidence for the assignment of structure was obtained by TLC followed by GC-MS analysis of the TMS derivative. A partial MS was presented and compared with that of a synthetic sample. Whereas cholestyramine treatment increases the levels of cyp7a activity in liver microsomes, subjects treated with the resin did not appear to show an increased level of 7-hydroxylase activity in liver microsomes for 26-OH-Chol. No conversion (limit of detection, 0.01 nmol·min¹·mg protein¹) of 26-OH-Chol to the ⁵-3,7,26-triol could be detected using human liver mitochondria. This finding differs from that made with pig liver mitochondria (35).

Axelson et al. (35) presented results indicating the first demonstration of the presence of 7-hydroxylase activity for 26-oxygenated sterols in liver mitochondria. Mitochondria from pig liver catalyzed the formation of 7-hydroxylated products from 26-OH-Chol and 3-hydroxycholest-5-en-26-oic acid. Products were characterized by GC-MS (TMS derivative of sterol and TMS derivative of the methyl ester of the acid). The combined results of this study indicated that the early reactions leading to the formation of bile acids can occur exclusively in mitochondria (i.e., not involving microsomal 7-hydroxylase). Toll et al. (1118) reported that pig liver microsomes catalyze the 7-hydroxylation not only of Chol but also of 26-OH-Chol, 3-hydroxycholest-5-en-26-oic acid, and 3-hydroxychol-5-enic acid. Two cytochrome P-450 species were isolated, one of which catalyzed the 7-hydroxylation of 26-OH-Chol, 3-hydroxycholest-5-en-26-oic acid, and 3-hydroxychol-5-enic acid but not of Chol. The other cytochrome P-450 species catalyzed the 7-hydroxylation of Chol and the other three substrates. The results were interpreted to "strongly indicate the presence of multiple microsomal sterol 7-hydroxylases." The same workers (976) also reported that human liver microsomes catalyzed the 7-hydroxylation of 26-OH-Chol and 3-hydroxycholest-5-en-26-oic acid. However, only slight activity was observed when Chol was used as the substrate. A P-450 fraction prepared from the microsomes efficiently catalyzed the 7-hydroxylation of 26-OH-Chol and 3-hydroxycholest-5-en-26-oic acid but had no detectable action on Chol. The 7-hydroxylation activity in microsomes for 26-OH-Chol was much higher in humans and pigs than in rats. The mitochondrial fraction of human liver also catalyzed the 7-hydroxylation of 3-hydroxycholest-5-en-26-oic acid. However, little or no 7-hydroxylation of 26-OH-Chol was observed. The 7-hydroxylation activity in mitochondria for 26-OH-Chol was much higher in pigs than in one human, rats, or rabbits. Human and pig mitochondria were also reported to catalyze the formation of 7-hydroxy products that were proposed to originate from epimerization of the corresponding 7-hydroxy compounds (through 7-keto intermediates). In a subsequent study, Toll et al. (1119) reported that liver microsomes from the rats, pigs, and humans catalyzed the 7-hydroxylation of 25-OH-Chol. Whereas treatment of rats with cholestyramine caused a marked increase in cyp7a activity (+281%) in liver microsomes, no significant (P > 0.05) effect of cholestyramine administration on the 7-hydroxylation of 25-OH-Chol was observed. Purification of Chol 7-hydroxylase from pig liver showed two 7-hydroxylase species, one for Chol and one for 25-OH-Chol (and 26-OH-Chol). The results indicated that at least 99% of the 7-hydroxylase activity toward 25-OH-Chol of pig liver microsomes was separated from the species catalyzing the 7-hydroxylation of Chol. Furthermore, transfection of the human liver cyp7a cDNA in COS cells showed significant cyp7a activity, whereas no significant 7-hydroxylation of 25-OH-Chol was observed.

Martin et al. (619) reported the 7-hydroxylation of 26-OH-Chol by microsomes of hamster liver, Hep G2 cells, and human liver. Little information was provided on the identification of the product. Under the specific assay conditions used in this study, the 7-hydroxylase activity for 26-OH-Chol in hamster liver microsomes was very much higher than that of Chol 7-hydroxylase, a finding also made with microsomes from human liver. In contrast, the 7-hydroxylase activity for 26-OH-Chol in microsomes from Hep G2 cells was less than that for 7-hydroxylation of Chol. Cholestyramine treatment of hamsters was reported to increase microsomal 7-hydroxylase activity for 26-OH-Chol (+56%) and cyp7a activity (+75%). The results of several types of experiments suggested that the microsomal enzyme responsible for 7-hydroxylation of 26-OH-Chol is different from cyp7a. Addition of cholestanol to hamster microsomes lowered cyp7a activity (50%) but had little (11%) effect on 7-hydroxylation of 26-OH-Chol. In contrast, 26-hydroxycholestanol lowered 7-hydroxylation of 26-OH-Chol (68%) but had little or no (20%; not significant) effect on cyp7a activity. Addition of a detergent, Emulgen 913, to hamster microsomes resulted in a differential lowering of activity; treatment with 0.2% detergent resulted in an ~70% lowering of 7-hydroxylase activity for 26-OH-Chol but only a ~30% lowering of cyp7a activity. Martin et al. (619) noted that they could detect no cyp7a activity or 7-hydroxylase activity for 26-OH-Chol in mitochondria preparations from hamster liver or Hep G2 cells. However, no data were presented on these points.

On the basis of studies of the metabolism of 26-OH-Chol and 7-OH-Chol to bile acids in Hep G2 cells (444), it has been suggested that 7-hydroxylation of 26-OH-Chol is not "well-expressed" in these cells. CHO-K1 cells have been reported neither to show cyp7a activity nor to contain detectable levels of mRNA for the 7-hydroxylase (263). Östlund Forrants et al. (743) reported that the level of cyp7a activity in Hep G2 cells is ~17% that observed in rat hepatocytes and very much less than that of rat liver microsomes.

Whereas 7-hydroxylation of Chol is commonly considered to occur exclusively in liver, the 7-hydroxylation of 26-hydroxylated and 25-hydroxylated sterols in other cell types has been reported. Zhang et al. (1225) reported the formation of 7-hydroxy derivatives upon incubation of 26-OH-Chol and 25-OH-Chol with human fibroblasts, indicating the potential extrahepatic formation of 7-hydroxysterols. Payne et al. (781) reported sterol 7-hydroxylase activity for 25-OH-Chol (but not Chol or testosterone) in isolated granulosa cells from rat ovary and in whole ovarian preparations which appears to be distinct from Chol 7-hydroxylase of liver. After incubation of ³H-labeled 25-OH-Chol with rat granulosa cells, two 7-hydroxylated metabolites were isolated and identified as cholest-5-ene-3,7,25-triol and 7,25-dihydroxycholest-4-ene-3-one on the basis of MS and ¹H-NMR data. The formation of these metabolites was markedly stimulated by interleukin-1. Incubations of [³H]Chol or [³H]testosterone with rat ovarian dispersates (in the presence or absence of interleukin-1) were reported to show no formation of 7-hydroxy metabolites. In addition, the authors were unable to detect any mRNA transcripts for liver cyp7a in rat ovarian dispersates. The interleukin 1-stimulated 7-hydroxylation of 25-OH-Chol was inhibited by the addition of unlabeled 25-OH-Chol or 26-OH-Chol. However, the addition of unlabeled Chol, pregnenolone, progesterone, testosterone, or dehydroepiandrosterone had no inhibitory effect on the 7-hydroxylation of 25-OH-Chol. Their combined results indicate the presence of a 7-hydroxylase for 25-OH-Chol in rat ovary which is distinct from cyp7a and steroid hormone 7-hydroxylase.

Stapleton et al. (1035) presented evidence for the existence of a novel cytochrome P-450 species in rat and mouse brain which, since it showed significant sequence homology with rat and human cyp7, was designated as cyp7b. Expression of cyp7b showed high specificity for brain, with only low levels of its expression in liver and kidney. Investigations of substrate specificity (861) indicated that dehydroepiandrosterone and pregnenolone underwent efficient 7-hydroxylation. 25-OH-Chol, the only C₂₇ sterol substrate studied, was converted to cholest-5-ene-3,7,25-triol. 7-Hydroxylation was also reported for 5-androstane-3,17-diol, 17-estradiol, testosterone, and progesterone; however, the reported yield for these 7-hydroxylated products was very much less than those for dehydroepiandrosterone and pregnenolone. No 7-hydroxylation was detected with corticosterone, cortisol, androstenedione, and dihydrotestosterone. Martin et al. (620) presented evidence indicating that cyp7b also catalyzes the 7-hydroxylation of 26-OH-Chol. They reported that transfection of CHO-K1 cells or 293/T cells with cDNA for mouse cyp7b led to the conversion of added 26-OH-Chol to cholest-5-ene-3,7,26-triol (on the basis of GC-MS analyses of the sterols recovered from the incubation medium). Little or no 7-hydroxylation of the 26-OH-Chol was detected in cells that were not transfected with cyp7b cDNA. Zhang et al. (1223) reported the 7-hydroxylation of 26-OH-Chol, 25-OH-Chol, and 3-hydroxycholest-5-en-26-oic acid, but not 24-OH-Chol, upon incubation with rat brain microsomal preparations. Schwarz et al. (942) observed that the 7-hydroxylation of 25-OH-Chol by mouse liver microsomes was inhibited by cholest-3,7,25-triol (IC₅₀ ~9 µM) but was not inhibited by 7-OH-Chol or cholest-5-ene-3,17,20-triol (at concentrations from 1.2 to 12 µM).

Schwarz et al. (943) reported transfection of human embryonic kidney 293 cells with an expression plasmid containing mouse cyp7b1 cDNA. Incubation of the transfected cells with [³H]25-OH-Chol led to the formation of labeled material with the TLC behavior of cholest-5-ene-3,7,25-triol and 7,25-dihydroxycholest-4-en-3-one. In addition, the formation of a third metabolite was detected, for which the structure cholest-5-ene-2,3,7,25-tetrol was suggested on the basis of the results of GC-MS of its TMS ether derivative and the reported formation of an acetonide derivative (data not provided). The authors concluded that the cyp7b1 cDNA was both an oxysterol 7-hydroxylase and a 2-hydroxylase. It is noteworthy that, in the same paper, studies of the metabolism of [³H]25-OH-Chol by liver microsomes (in the presence of NADPH) from a number of species (including human) appear to show little or no radioactive metabolite with the mobility of the 2,3,7,25-triol. Chol feeding (2% in diet for 10 days) to male mice was reported to have no effect on the level of cyp7b mRNA in liver and little or no effect on cyp7b enzyme activity in liver microsomes (assayed by TLC with [³H]25-OH-Chol as substrate) or on hepatic oxysterol 7-hydroxylase activity. Administration of colestipol (2% in diet) had little or no effect on hepatic cyp7b mRNA, enzyme activity, or enzyme protein. Feeding cholic acid (0.5% in diet) lowered cyp7b mRNA in liver (approximately 70%); however, this reduction was less than that observed for cyp7a mRNAs (i.e., almost complete absence). Cholic acid administration was associated with ~40% lowering of cyp7b enzyme activity and enzyme protein. A limitation of these studies was the use of pooled samples of liver from individual mice (n = 4 or 5), thus precluding any statistical evaluation of the experimentation.

Norlin and Wikvall (717) recently reported that pig liver microsomes catalyzed the 7-hydroxylation of 26-OH-Chol and 25-OH-Chol as well as dehydroepiandrosterone and pregnenolone. Chol and testosterone were also 7-hydroxylated but to a very low extent. These authors reported on extensive efforts to determine if the cytochrome P-450 in the pig microsomes for 7-hydroxylation of 26-OH-Chol was the same as that catalyzing the 7-hydroxylation of dehydroepiandrosterone. An extensively purified enzyme catalyzed the 7-hydroxylation of 26-OH-Chol, 25-OH-Chol, dehydroepiandrosterone, and pregnenolone. With the purified enzyme, no 7-hydroxylation of Chol or testosterone was observed. No separation of activities for the 7-hydroxylation of 26-OH-Chol and dehydroepiandrosterone was observed during the purification. However, notably large variations were observed in the ratio of 7-hydroxylation of the two substrates in different experiments. Studies with five inhibitors indicated no selective effect on the 7-hydroxylation of the two substrates. With the purified enzyme, addition of 26-OH-Chol had no inhibitory effect on the 7-hydroxylation of dehydroepiandrosterone. However, dehydroepiandrosterone inhibited the 7-hydroxylation of 26-OH-Chol. Interestingly, the inhibition was noncompetitive, suggesting, if one enzyme is involved, the possibility of the presence of more than one active site. In summarizing their studies the authors favored the idea that two enzymes are involved in the 7-hydroxylation of the two substrates.

Post et al. (815) reported that cafestol, a diterpene responsible for the hypercholesterolemic effect of boiled coffee, caused a dose-dependent decrease in cyp7a activity, bile acid synthesis, and cyp26 activity in cultured rat hepatocytes. The decrease in 7-hydroxylase activity was associated with decreases in its mRNA levels and in cyp7a gene transcriptional activity, which were observed at concentrations as low as 16 and 32 µM, respectively.

Stravitz et al. (965) reported that taurine conjugates of cholic acid and deoxycholic acid caused a concentration-dependent lowering of the levels of mRNA for cyp7a in primary cultures of rat hepatocytes. In contrast, the taurine conjugates of ursodeoxycholic acid, hyodeoxycholic acid, and ursocholic acid had no effect under the same study conditions. Twisk et al. (1139) found that either deoxycholic acid or cholic acid (at 50 µM) caused a marked lowering of cyp7a activity, mRNA levels, and transcriptional activity in rat hepatocytes. Similar findings were made with cyp26. Little or no effects were observed with two hydrophilic bile acids, -muricholic acid and ursocholic acid. The results of studies of the effects of 27 different bile acids (at 50 µM) were presented. A moderate correlation between the hydrophobicity of the bile acids and cyp7a mRNA levels was observed. Pandak et al. (754) reported that, in Hep G2 cells, the hydrophobic bile salts, glycochenodeoxycholate and glycodeoxycholate, decreased bile acid synthesis and cyp7a mRNA levels in a time-dependent and concentration-dependent fashion. The hydrophilic bile salts, glycoursodeoxycholate and glycohyodeoxycholate, had no effect on cyp7a mRNA levels nor did glycocholate (intermediate hydrophobicity).

Whereas certain hydrophobic bile acids have striking effects in downregulating cyp7a in cultured hepatocytes, the regulation of cyp7a by bile acids in animals appears to be much more complicated. For example, Pandak et al. (752) observed that intraduodenal administration of taurocholate to bile fistula rats resulted in a clear decrease in hepatic cyp7a activity, mRNA levels, and transcriptional activity. However, intravenous administration of taurocholate had no effect on cyp7a. An intestinal factor, as yet unidentified, was proposed as a possible important regulatory species of cyp7a to account for this discrepancy.

Stavitz et al. (1049) reported findings indicating that protein kinase C is involved in the lowering of cyp7a gene transcription in primary cultures of rat hepatocytes caused by hydrophobic bile acids. Activation of protein kinase C with phorbol 12-myristate 13-acetate (0.1 µM) decreased mRNA for cyp7a (71%) and transcriptional activity (60%). Taurocholate (25 µM) decreased mRNA for the 7-hydroxylase. However, this effect of taurocholate could be blocked by preincubation of the cells with inhibitors of protein kinase C. Further studies showed that hydrophobic bile acids caused an increase in the amount of protein kinase C that was associated with membranes. Stravitz et al. (1050) extended these studies. Nonselective activation of protein kinase C isoforms with phorbol 12-myristate 13-acetate resulted in a 75% decrease in cyp7a mRNA levels in rat hepatocytes. However, thymeleatoxin, a phorbol compound reported to selectively activate calcium-independent protein kinase C isoforms, had little effect on cyp7a mRNA levels in rat hepatocytes. These and other results led to the suggestion that calcium-independent protein kinase C isoforms are involved in the repression of cyp7a gene transcription by taurocholate.

Crestani et al. (222) reported that phorbol 12-myristate 13-acetate, an activator of protein kinase C, caused an inhibition of cyp7a promoter activity in Hep G2 cells transfected with rat cyp7a promoter/luciferase chimeric genes. The location of the negative phorbol ester response sequences were mapped. In contrast, the levels of 7-oxygenated sterols (7-OH-Chol, 7-OH-Chol, and 7-keto-Chol) as well as those for 5,6-epoxy-Chol, 5,6-epoxy-Chol, and 20-OH-Chol in U 937 cells (a monocyte-like cell line) were reported to increase upon incubation with phorbol 12-myristate 13-acetate (100 ng/ml) for 60 min. The reported formation of the 20-OH-Chol is noteworthy. However, it is important to note that characterization of the oxysterols was limited to chromatographic behavior on capillary GC of their TMS derivatives.

Taniguchi et al. (1094) studied the expression of the cyp7a gene in Hep G2 cells. Incubation of the cells in complete medium (containing 10% FCS) for 2, 4, 8, and 24 h was reported to show a gradual increase in the levels of mRNA for cyp7a with time with a maximum twofold increase at 24 h. However, considerable experimental variation was observed, and significant (P < 0.05) elevation of mRNA was observed only at 2 h. The claimed increase in mRNA levels under these conditions appeared to be blocked or suppressed by the addition of very-low-density lipoprotein (VLDL; 40 µg/ml) or 25-OH-Chol (12.4 µM). Similar incubations of the Hep G2 cells in serum-free medium was reported to result in substantial and significant (P < 0.05) increases in the levels of mRNA for cyp7a at all time points studied (i.e., 2, 4, 8, and 24 h) with a maximum increase (~5-fold) at 8 h. These substantial increases in mRNA levels in cells cultured in serum-free medium were blocked or suppressed by the addition of VLDL (40 µg/ml) or 25-OH-Chol (12.4 µM). The results of studies of the transcriptional activity for the cyp7a gene with nuclei from Hep G2 cells (grown in serum-free or complete media for 8 h) indicated that the increased levels of mRNA appeared to be the result of transcriptional regulation.

Taniguchi et al. (1094) also reported that dexamethasone (1 µM) caused an increase in the levels of mRNA for cyp7a in Hep G2 cells grown in serum-free medium. Progesterone (32 µM) was reported to decrease the levels of mRNA for the 7-hydroxylase. The same authors reported that chenodeoxycholic acid caused a dose-dependent decrease in the levels of cyp7a mRNA in Hep G2 cells grown in serum-free medium for 8 h. Chenodeoxycholate (100 µM) caused significant lowering as early as 2 h. Nuclei from cells incubated with this bile acid (100 µM) for 8 h were reported to show a lowering of cyp7a gene transcription. Ramirez et al. (835) also reported results indicating transcriptional control of cyp7a in a transformed hepatocyte cell line and in transgenic mice. Hylemon et al. (420) studied the hormonal control of the cyp7a mRNA levels and of transcriptional activity of the cyp7a gene in cultures of adult rat hepatocytes. The combination of dexamethasone (0.1 µM) and thyroxine (1.0 µM) resulted in a marked increase in the levels of cyp7a mRNA, whereas the individual hormones, at the same concentrations, had relatively little effect. The individual hormones increased the levels of transcriptional activity for cyp7a, and the combination of the two hormones resulted in a marked increase. Further hormonal control of the 7-hydroxylase was indicated by the lowering of mRNA levels of either glucagon (0.2 µM) or dibutyryl cAMP (50 µM). Taurocholate lowered the levels of cyp7a mRNA in rat hepatocytes incubated in the presence of added thyroxine and dexamethasone, with an IC₅₀ of ~25 µM (420). Trawick et al. (1122) reported that dexamethasone (100 µM) increased cyp7a mRNA levels in rat hepatoma (L35) cells and that the increase caused by dexamethasone was augmented by the addition of dithiothreitol (DTT) or reduced glutathione. In the absence of the steroid, DTT or reduced glutathione had no effect on cyp7a mRNA levels. Dexamethasone plus DTT increased cyp7a transcriptional activity, and DTT reversed the repression of cyp7a transcription caused by insulin but not that caused by phorbol 12-myristate 13-acetate. The combined results were interpreted as indicating that the level of reduced glutathione has an important influence on the transcription and expression of the cyp7a gene.

Story et al. (1047) reported that administration of either D-thyroxine or L-thyroxine to rats elevated the levels of hepatic microsomal cyp7a activity, whereas propylthiouracil depressed the level of hepatic microsomal activity. However, when the results were expressed on the basis of enzyme activity per liver, no significant effects of the administration of D-thyroxine, L-thyroxine, or propylthiouracil were observed. Ness et al. (704) recently reported that intraperitoneal injection of L-triiodothyronine (100 µg/100 g body wt) to hypophysectomized male rats resulted in a very substantial increase (8-fold) in the levels of cyp7a mRNA at 6 h. Timed studies carried out after administration of L-triiodothyronine (100 µg/100 g body wt) indicated a maximal stimulation at 1 h. Hepatic microsomal cyp7a activity was also reported to increase and to reach a maximum at 90 min; however, no data were presented on this point. Increased levels of cyp7a mRNA in livers of thyroid hormone-deficient rats have been observed after administration of thyroid hormone (704, 705). The same authors (703) have reported that administration of triiodothyronine to thyroidectomized rats increases transcription of the cyp7a gene by liver nuclei. However, in humans with hypothyroidism, administration of thyroid hormone (either triiodothyronine or thyroxine) had no effect on the levels of 7-hydroxycholest-4-en-3-one (an indirect measure of hepatic cyp7a activity) in serum despite the observation of significant decreases in LDL Chol and total Chol in serum (902). Medical treatment of hyperthyroidism resulted in significant increases in LDL Chol and total Chol in serum; however, no significant change in the levels of 7-hydroxycholest-4-en-3-one was observed. Thus changes in thyroid hormone status, sufficient to modify Chol levels in serum, had no effect on hepatic cyp7a activity (at least as studied by effects on the levels of the 7-hydroxy-3-ketosterol in serum).

Daily treatment of rats for 4 days with dexamethasone (100 mg/kg) or pregnenolone-16-carbonitrile (50 mg/kg) by intraperitoneal injection in corn oil resulted in a very marked lowering of the level of cyp7a activity in liver microsomes (197). No effect of phenobarbital (100 mg/kg) administration was observed. Glucocorticocoids (dexamethasone, cortisol, and corticosterone), but not other steroid hormones (17-ethinylestradiol, progesterone, testosterone, androsterone, pregnenolone, and aldosterone), have been reported to increase the conversion of [¹⁴C]Chol to bile acids in cultured rat hepatocytes (822). Dexamethasone (1 µM) had no effect on cyp7a activity at 24 h; however, very large increases were observed at 48 and 72 h. In contrast, treatment of rats with dexamethasone (100 mg/kg) or with pregnenolone-16-carbonitrile was reported to markedly reduce the levels of cyp7a mRNA in liver (564) and to lower the levels of enzyme activity and protein. Data on the latter two points were not presented. However, it should be noted that earlier studies reported a doubling of cyp7a activity in rat liver at 3 h after the intraperitoneal administration of synthetic or natural corticosteroids (1151). The diurnal rhythm for the 7-hydroxylase in rats was also obliterated by hypophysectomy or adrenalectomy (335). Administration of either conjugated equine estrogen or medroxyprogesterone acetate has been reported to increase the levels of hepatic cyp7a mRNA in ovariectomized adult female cynomologus monkeys (212).

In studies of the hormonal regulation of cyp7a, Crestani et al. (222) reported that insulin inhibited cyp7a promoter activity in Hep G2 cells transfected with rat cyp7a promoter/luciferase chimeric genes. In the same study, a glucocorticoid (dexamethasone) and all trans-retinoic acid activated cyp7a promoter activity. In the same system, thyroxine had no effect on cyp7a promoter activity. Crestani et al. (223) reported the identification of a hormone response unit for cyp7a promoter activity that mediates the opposing effects of retinoic acid and phorbol 12-myristate 13-acetate. The activation of promoter activity was observed with both all trans-retinoic acid and 9-cis-retinoic acid. Sadeghpour et al. (880) reported identification of the portion of the sequence responsible for the effects of retinoic acid and phorbol 12-myristate 13-acetate (as well as insulin) and that mutations in this region resulted in the elimination of the effects of retinoic acid, phorbol 12-myristate 13-acetate, and insulin and a decrease in the effect of dexamethasone. Two bile acid response elements in the promoter region of the rat cyp7a gene have recently been identified (199, 1056). Foti and Chiang (311) reported findings indicating that a transcriptional factor [basic transcription element-binding protein (BTEB)] binds to a region corresponding to a bile acid response element of the rat cyp7a promoter and that overexpression of BTEB resulted in a repression of transcriptional activity of cyp7a.

Twisk et al. (1138) reported that insulin, at physiological concentrations, inhibited the formation of bile acids and lowered cyp7a activity. The decrease in enzyme activity caused by insulin was associated with a decrease in the level of mRNA for the enzyme and decreased transcriptional activity. As noted previously, very similar results were observed with regard to the effects of insulin on cyp26.

Rudling et al. (875) reported findings indicating the importance of growth hormone on hepatic cyp7a. Hypophysectomized rats showed lowered levels of enzyme activity (64%). Interestingly, the levels of hepatic cyp7a mRNA were increased (+81%). It was proposed that a deficiency of growth hormone as a consequence of hypophysectomy results in decreased hepatic cyp7a activity, which in turn leads to a decreased synthesis and fecal excretion of bile acids and that the resulting decreased levels of bile acids in liver caused a derepression of the transcription of the cyp7a gene with resulting increased levels of cyp7a mRNA. Rudling et al. (875) also reported that infusion of growth hormone to hypophysectomized rats resulted in restoration of the levels of hepatic cyp7a activity to normal levels. Administration of growth hormone to normal rats fed a diet enriched in Chol (2%) and sodium cholate (0.5%) was associated with a marked increase in the levels of hepatic cyp7a activity.

Administration of Chol or of cholestyramine increases the levels of cyp7a activity, its mRNA, and transcriptional activity in rat liver, whereas feeding of bile acids, in general, reduces these parameters (449, 564, 753, 755, 756). However, feeding of deoxycholic acid at a high level (1% in diet for 10 days) not only did not result in a decrease in cyp7a mRNA levels but caused a very substantial increase in the levels of mRNA for this protein (513). A large fraction of this increase appeared to be related to decreased food consumption (associated with decreased body weight). The increase in mRNA levels by deoxycholic acid feeding appeared to be largely due to posttranscriptional processing.

Several studies (753, 755, 965, 1137) have indicated repression of cyp7a by taurocholate at the level of gene transcription. Pandak et al. (756) reported that feeding of cholic acid (1% in diet), chenodeoxycholic acid (1% in diet), or deoxycholic acid (0.25% in diet) to male rats resulted in marked reduction of the levels of enzyme activity, mRNA, and transcriptional activity for cyp7a in liver. The levels of specific activity of the 7-hydroxylase were suppressed more than the levels of transcriptional activity, leading to the suggestion of additional posttranscriptional regulation. Shefer et al. (966) reported that feeding deoxycholic acid (0.4 or 1.0% in diet) to male rats resulted in a marked lowering of cyp7a activity in liver, which was associated with a very marked increase in the level of mRNA for the enzyme. In contrast, deoxycholate was reported to have no effect on the levels of hepatic 27-hydroxylase activity or on the levels of mRNA for this enzyme. Administration of cholestyramine (5% in diet) to male rats resulted in a marked elevation of the levels of activity, mRNA, and transcriptional activity for cyp7a in liver as well as an elevation of the levels of HMG-CoA reductase activity. In another study (318), cholestyramine administration (2% in diet for 2 wk) to male rats was associated with a substantial increase in the levels of hepatic microsomal cyp7a activity and 7-OH-Chol in serum. Horton et al. (400) reported that dietary administration of psyllium (7.5% in diet) or cholestyramine (1% in diet) to hamsters resulted in increases in the levels of hepatic cyp7a activity and mRNA. Hayashi et al. (377) reported that administration of cholestyramine (2% in diet for 14 days) to Syrian golden hamsters resulted in 600% increase in the levels of cyp7a activity in liver.

Maeda et al. (601) observed that the levels of cyp7a activity and protein (determined by immunochemical assay) were higher in liver microsomes from patients (n = 3) treated with cholestyramine than in those from untreated patients (n = 6). However, increases in enzyme activity and enzyme content were not proportional. In the treated patients, the levels of enzyme protein in liver microsomes were reported to be "approximately twofold higher than those of liver microsomes from untreated patients" (P < 0.02). However, the level of enzyme activity was "approximately sixfold higher" in liver microsomes from patients treated with the resin than the activity in microsomes from untreated patients. These findings were interpreted as suggesting, in addition to transcriptional control, "a posttranslation mechanism."

Sudjana-Sugiaman et al. (1062) introduced the cDNA for human cyp7a into COS cells and observed the production of 7-hydroxylase protein and the accumulation within the cells of 7-OH-Chol. The mean level of cyp7a activity in the transfected cells was 0.26 ± 0.05 pmol·min¹·mg protein¹ (cf. 0.03 ± 0.02 in control cells). The transfected cells showed low but significant levels of 7-OH-Chol (from 11 to 67 ng/mg cell protein). A high correlation between the level of cyp7a activity in the transfected cells and the basal level of 7-OH-Chol in the cells was reported. The levels of 7-OH-Chol were based on GC-MS methodology (although no characterization data were presented). It was stated that no 7-OH-Chol was detected. The level of HMG-CoA reductase activity was reported to be higher (+58%) than that in control cells. The free Chol levels in the transfected COS cells were not different from control values.

A number of investigations have indicated that cyp7a activity can be modified in vitro by phosphorylation-dephosphorylation processes (209, 214, 256, 347, 397, 530, 709, 893, 903, 1093). Other investigators (60, 274, 578) have not obtained results consistent with this potential in vitro modulation of cyp7a activity, perhaps due to differences in experimental conditions (709). Whereas phosphorylation-dephosphorylation changes of the enzyme may modulate cyp7a in vitro, this author is unaware of any studies demonstrating the importance of such mechanisms in the control of cyp7a activity in intact cells or in vivo in animals. Cytosolic proteins have been reported to affect cyp7a activity in vitro (237, 238, 529 and references cited therein). Danielsson et al. (238) purified a protein (M_r 25,000) from rat liver cytosol that stimulated cyp7a activity. Stimulation was observed only in the presence of reduced glutathione or reduced thioredoxin. The activation of cyp7a activity by the cytosolic protein did not appear to be affected by ATP, MgCl₂, or sodium fluoride.

Bensch et al. (59) reported that acute (3 or 24 h) oral administration of ML-236B (compactin; 20 mg/kg) to male Sprague-Dawley rats had no significant effect on the levels of cyp7a activity in liver. Endo et al. (281) found that oral administration of ML-236B twice a day at 250 mg/kg for 11 days to male Wistar-Imamichi rats caused a modest (31%) reduction in the levels of cyp7a activity in liver and a decrease (53%) in fecal bile acids. Björkhem (82) reported that dietary administration of mevinolin (0.1% in a "commercial fat-free diet") for 3 days to male Sprague-Dawley rats caused a decrease (36%) in the levels of cyp7a activity when assayed for the 7-hydroxylation of exogenous labeled Chol. However, no significant change in cyp7a activity was observed when the enzymatic activity was assayed by determination of the 7-hydroxylation of endogenous Chol in liver microsomes. Mevinolin administration had no effect on the levels of free Chol in liver microsomes. Björkhem (86) reported that dietary administration of mevinolin (0.1% in diet) to male Syrian golden hamsters for 6 days had no significant effect on hepatic cyp7a activity. They also reported that dietary administration of mevinolin (0.1%) to male Sprague-Dawley rats decreased the rise in hepatic cyp7a activity induced by the administration of cholestyramine (5% in diet) or Chol (2% in diet). More recently, Jones et al. (458) reported that intravenous administration of mevinolin to rats with chronic biliary cannulation resulted in lowering of liver microsomal cyp7a specific activity and a lowering of enzyme mass, mRNA, and transcriptional activity for the 7-hydroxylase. In contrast, intravenous administration of mevalonate resulted in increases in cyp7a specific activity, mRNA levels, and transcriptional activity. The same laboratory (1173) also reported that short-term biliary diversion in rats resulted in a substantial increase in cyp7a (+259% at 48 h and +827% at 96 h). Treatment with mevinolin prevented the increase in 7-hydroxylase activity induced by the biliary diversion. In contrast, administration of mevalonic acid caused a marked increase in cyp7a activity over that induced by biliary diversion alone. Under the conditions of this study, i.e., short-term biliary diversion, the results suggested that regulation of the 7-hydroxylase was under the control of both "bile acids and newly synthesized Chol or its metabolites" (which were suggested to be oxysterols). Atorvastatin, an inhibitor of HMG-CoA reductase, had no significant effect on hepatic microsomal cyp7a activity after dietary administration of the drug in a high-fat diet to guinea pigs for 3 wk (213). Pravastatin, another competitive inhibitor of HMG-CoA reductase, had no effect on the levels of cyp7a activity in livers of Syrian golden hamsters under the conditions studied (377). Pravastatin administration to normal male subjects had little or no acute effect on bile acid synthesis, at least as indicated by its effects on the levels of 7-hydroxycholest-4-en-3-one in plasma (1217).

Zaragozic acid A (squalestatin 1), a squalene synthetase inhibitor, at a concentration of 1 µM, caused a very marked lowering (~98%) of cyp7a activity and a marked lowering of steady-state mRNA levels for the 7-hydroxylase in primary cultures of rat liver cells (258). Direct addition of squalestatin 1 (150 µM) to liver microsomes (from male rats fed a diet containing cholestyramine) had no effect on cyp7a activity (258). The marked lowering of cyp7a activity and mRNA levels by squalestatin 1 in rat hepatocytes was reversed by the addition of Chol (200 µM in -cyclodextrin). The effect of administration of zaragozic acid A on the levels of liver mRNA for cyp7a has been studied in rats (706). Subcutaneous administration of zaragozic acid (1, 2, or 5 mg/kg body wt 9 h before death) was reported to result in decreases in the levels of cyp7a mRNA. In the same experiment, substantial increases in mRNA for the LDL receptor were observed. The levels of mRNA in liver for HMG-CoA reductase, squalene synthetase, and HMG-CoA synthase increased (with maximum values observed at ~6 h) after a single subcutaneous injection of zaragozic acid A.

Bezafibrate administration (200 mg 3 times/day for 4 wk) to human subjects caused a lowering (~60%) of the levels of cyp7a activity in liver (1033).

Administration of phenobarbital to different strains of rats has been reported to give variable acute effects on the levels of cyp7a, with increases in some strains and decreases or no changes in others. Sudjana-Sugiaman et al. (1061) reported that three of nine rat strains (Wistar F, Lewis, and Brown Norwegian) showed increases of cyp7a activity in liver microsomes after daily intraperitoneal administration of phenobarbital (100 mg/kg body wt). The increases in 7-hydroxylase activity were maximal at 48 h and decreased to control levels at 96 h. In one typical set of experiments, the specific activity of cyp7a was 62 ± 13 pmol·min¹·mg protein¹ in Wistar F rats treated with phenobarbital for 48 h and 29 ± 5 pmol·min¹·mg protein¹ in the corresponding control rats. The levels of mRNA for cyp7a also showed increases that appeared to be maximal at 48 h. It is interesting to note that whereas the Wistar F, Lewis, and Brown Norwegian rats showed increases in cyp7a activity after phenobarbital administration, three other rat strains showed decreases in 7-hydroxylase activity after phenobarbital administration for 48 h. Gerbils and three other rat strains showed no significant changes in cyp7a activity under the same conditions. Strain differences in hepatic cyp7a activity have also been reported in mice. C57BL/6 mice (a strain reported to have increased susceptibility to the development of atherosclerosis on a high-Chol diet) were reported to show higher levels of hepatic cyp7a activity and mRNA than BALB/c mice when the animals were on a chow diet (264). Administration of a high-Chol diet increased cyp7a activity in both strains. Addition of taurocholate to the high-Chol diet markedly lowered cyp7a activity, significantly more so in the C57BL/6 mice. Kirk et al. (494) reported a high degree of variation in the levels of hepatic cyp7a mRNA in different strains of mice. Moreover, different strains of mice showed different effects of feeding safflower oil or safflower oil plus Chol on the levels of cyp7a mRNA in liver. Three of nine strains showed increased levels upon safflower oil administration in a rodent chow diet. One of the nine strains showed a significant decrease. Feeding of Chol plus safflower oil in the rodent chow diet showed a further increase (relative to safflower oil diet alone) in one strain. One strain of mice showed no change in cyp7a mRNA on either the safflower oil diet or the safflower oil plus Chol diet. Poorman et al. (814) reported increased levels of cyp7a activity in livers of an inbred strain of NZW rabbits in which there was little or no elevation of serum Chol levels upon Chol feeding. The Chol-resistant rabbits also showed increased levels of cyp7a mRNA in liver (relative to that observed in normal rabbits). The increased level of the 7-hydroxylase activity (and of mRNA) correlated with the increase in excretion of bile acids observed in the Chol-resistant rabbits upon Chol feeding (relative to that observed in normal rabbits).

Dietary administration of 7-keto-Chol (0.1% in chow diet) to rats for 6 days resulted in an increase (+170%) in the levels of liver microsomal cyp7a activity (1091). Administration of the 7-ketosterol was associated with significant decreases in the levels of free Chol (30%), Chol esters (64%), and total Chol (34%) in liver microsomes and marked increases in the levels of 7-keto-Chol (+771%) and 7-OH-Chol (+2,922%) in liver microsomes. No significant effect on the level of 7-OH-Chol in microsomes was observed. The increased hepatic microsomal cyp7a activity after dietary administration of 7-keto-Chol as reported by Tamasawa et al. (1091) is in contrast to reported in vitro inhibitory effects of 7-keto-Chol on cyp7a activity of liver microsomes (965, 1152). Doerner et al. (258) studied the effects of the addition of several oxysterols on the levels of cyp7a mRNA in primary rat hepatocytes. 5-Cholestane-3,6-diol, but not 7-OH-Chol, 5-cholestane-3,5,6-triol, or (25R)-26-OH-Chol, had a significant effect (an increase) on the levels of cyp7a activity. The oxysterols were added in 2.5% -cyclodextrin at a single concentration (not specified). None of the above oxysterols (at 50 µM; added in -cyclodextrin) reversed the effect of squalestatin 1 (a marked lowering of cyp7a activity). Feeding an oxysterol mixture (0.5% in a Chol-free diet) resulted in lower levels of cyp7a activity in livers of male rats (735). A limitation of this study was the lack of a pair-fed control group to deal with the decreased food consumption caused by the administration of the oxysterol mixture. Feeding the same mixture of oxygenated derivatives of Chol to rats at a level of 0.2% in a basal Chol-free diet for 20 days lowered the level of cyp7a activity in liver (739). Osada et al. (740) reported on the levels of cyp7a activity in livers from 4-wk-old rats fed one of three diets: a basal Chol-free diet, the basal diet containing added Chol (0.5%), or the basal diet supplemented with Chol (0.5%) and a mixture of oxidized Chol species (0.5%). The major component in the oxysterol mixture was 7-keto-Chol (22%), which was accompanied by a number of other Chol oxidation products and unidentified material. Because administration of the diet supplemented with Chol plus the oxidized Chol mixture suppressed food consumption (data not shown), the animals receiving the basal diet or that supplemented with Chol were given food only in the amount consumed by the animals receiving the diet supplemented with both Chol and the oxidized Chol mixture. The levels of cyp7a activity in liver were elevated in the Chol-fed animals. This elevation was partially blocked by the inclusion of the oxidized Chol mixture in the Chol-supplemented diet.

Bile duct ligation has been reported to elevate cyp7a activity in rat liver (236, 364). Selective portal vein ligation did not affect the levels of 7-hydroxylase activity in liver of male Wistar rats (608). In these studies, the branch of the portal vein supplying the left lateral and median lobes of the liver was ligated, and liver samples from the left lateral and right lobes were obtained for analysis of cyp7a activity. On the basis of these and other findings, the authors concluded that the intrahepatic concentrations of Chol and bile acids had no regulatory effect on cyp7 under the conditions studied.

Shefer et al. (967) reported decreased levels (~39% lower than those of control subjects) of cyp7a activity in liver microsomes of human subjects with sitosterolemia. Assay of cyp7a activity using acetone-treated microsomes (to remove endogenous sterols) showed no difference in cyp7a activity between the sitosterolemic and control subjects. The levels of hepatic HMG-CoA reductase activity were markedly lower in the human subjects with sitosterolemia (relative to controls).

The levels of cyp7a undergo dramatic developmental changes in the rat (999). Very low levels ("near the limits of detectability") of cyp7a activity were observed in fetal liver (1 or 4 days before birth). By 18 h after birth, the level of 7-hydroxylase activity "increased to about 40% of the adult level." The levels of enzyme activity then fell so that on days 3 and 6 the activity was almost undetectable. The values then increased [days 12, 17, 18, 19, 21 (weaning), and 28]. The highest value was on day 21. In general, the changes in the levels of cyp7a protein followed that of activity except for the day 1, day +1 (18 h) values when the protein values were very much higher than the values for enzyme activity. Day 1 (18 h) to day 19 corresponded to suckling, and day 21 corresponded to weaning. Oren et al. (733) reported that hepatic microsomal cyp7a activity in piglets was not detectable (<1.0 pmol·min¹·mg protein¹) during gestation or at birth. 7-Hydroxylase activity increased after birth to 6.8 ± 2.6 pmol·min¹·mg protein¹ at 3 wk and 18.2 ± 2.5 pmol·min¹·mg protein¹ at 7 wk. Fasting (12 h) decreased cyp7a activity. Bertolotti et al. (71) found a lower level of 7-hydroxylation in older human subjects (above 62 yr). The total study included 18 males and 10 females in which 7-hydroxylation was assayed by a method involving a tritium-release assay after intravenous injection of [7-³H]Chol. Spady et al. (1022) reported that, in short-term experiments (2 days), infection of hamsters with a recombinant adenovirus encoding rat cyp7a increased microsomal cyp7a activity in liver and a lowering of plasma total Chol and LDL Chol. The magnitudes of these changes were related to the amounts of the administered recombinant adenovirus. The reduction in LDL Chol levels could also be affected, in short-term experiments, by overexpression of cyp7a in LDL receptor / mice (1024). Moore et al. (670) reported the expression of human cyp7a in atherosclerosis-susceptible mice using an adenovirus vector. At 2 wk after infection, mice receiving the adenovirus vector containing the human cyp7a cDNA showed total plasma Chol and LDL-VLDL Chol levels that did not appear to differ from those of mice infected with the adenovirus vector alone. Partial transfection of liver with a synthetic cyp7a gene using a nonviral delivery vector has been reported to affect plasma Chol levels in mice in short-term experiments (5).

Ishibashi et al. (426) reported the production of a mutant strain of mice affecting cyp7. Newborn homozygous (Cyp7 /) mice appeared normal at birth but showed high mortality (~85%) in the first 18 days of life. The mortality appeared to occur in two phases, one in the period from days 1 to 4 that could be suppressed by the addition of a vitamin mixture to the drinking water of nursing mothers and another in the period from days 11 to 18 that could be suppressed by the addition of cholic acid to the chow consumed by nursing mothers. Liver microsomal preparations from the Cyp7 / mice showed no cyp7a activity. The mutant mice born to Cyp7 / mothers fed normal chow showed severely depressed gain in body weight, indications of lowered liver cytochrome P-450 content, elevated levels of conjugated bilirubin in liver, lowered motor activity, and changes in skin (dry scaly appearance accompanied by thickening of stratum corneum and granular layers) and in eye (delayed opening). Schwarz et al. (942) obtained additional results on this important mutant mouse. Stool fat levels of Cyp7 +/+ mice were clearly elevated during the first 3 wk of life but by 4 wk returned to the level of wild-type mice. The mutant mice showed suggestions of lowered levels of vitamin D in serum on days 6, 16, and 23 (based on analyses of pooled serum samples from mutant and wild-type groups). Little or no differences between mutant and wild-type mice were observed on days 34-38 and 120-180. In contrast to comments by the authors, provision of a vitamin mixture appeared to have no consistent effects on the levels of vitamin D in serum. The levels of vitamin E in samples of fat (epididymal or ovarian fat pads) appeared to be clearly lowered in mutant mice (relative to wild-type animals) on days 16, 23, 34-38, and 120-180. Supplementation with a vitamin mixture appeared to have no consistent effects on the levels of vitamin E. Supplementation with vitamins and cholic acid increased the levels of vitamin E in the fat pads of the mutant mice. It is important to note that the levels of vitamin E in fat samples were expressed relative to triglyceride and that information of the effects of the mutation on epididymal or ovarian triglyceride levels in the ovarian and epididymal fat pads was not presented. The mutant mice showed little or no differences from wild-type animals in the levels of Chol and triglycerides in serum at various points of development (6, 15, 23, 60-90, and 150-180 days). Analyses of bile acid composition and levels in bile and feces from adult Cyp7 / mice and wild-type mice were also reported. The concentration of total bile acids in bile did not appear to differ in the mutant and wild-type mice (although the analyses were limited to two each of the mutant and wild-type mice). However, lower levels of total bile acids in feces from the mutant mice were reported. Cholic acid, the major bile acid in bile of wild-type mice, appeared to be reduced in the mutant. In contrast, chenodeoxycholic acid, -muricholic acid, and hyodeoxycholic acid were not detected in bile from wild-type animals but were present at significant levels in bile from the Cyp7 / mice. As noted, the mean level of total fecal bile acids in the mutant mice was lower than that of wild-type mice. -Muricholic acid (3,6,7-trihydroxy-5-cholanic acid), which was not detected in feces from any of the mutant mice, was present at significant levels in feces from wild-type animals. This is in notable contrast to the case of bile, in which this acid was not detected in wild-type mice but was present at substantial levels in the bile from mutant animals. An important finding was the observation of substantial levels of 7-hydroxylated bile acids in the Cyp7 / mice. A possible explanation for the occurrence of the 7-hydroxy bile acids in the mutant mice was provided by the demonstration of the presence of 7-hydroxylase activity for 25-OH-Chol in liver microsomes of the mutant mice. The product was characterized by its TLC and by MS (data not shown). Very low levels of the enzyme activity were present through the first 3 wk of life in both mutant and wild-type animals. Thereafter, the levels of activity increased substantially in both mutant and wild-type mice. The induction of this activity corresponded to the timing of the decrease of elevated levels of fecal fat in the mutant mice. The authors considered that the occurrence of substantial levels of the 7-hydroxylated bile acids in the adult mutant mice arose from an alternative mode of bile acid formation not involving initial 7-hydroxylation of Chol but involving initial mitochondrial hydroxylation to give 26-OH-Chol followed by its 7-hydroxylation via the induced (at ~4 wk) microsomal oxysterol 7-hydroxylase and then subsequent metabolism of the 3,7,26-trihydroxysterol to bile acids. Schwarz et al. (943) subsequently reported on the developmental course of the levels of cyp7b mRNA, protein, and enzyme activity (assayed by TLC with [³H]25-OH-Chol as substrate) in wild-type mice. After appearance at about day 18, the levels of each of the above increased thereafter. Assays were made on pooled samples obtained from livers of mice (3-10/time point). The combined results with the mutant Cyp7 / mice demonstrate the critical importance of cyp7a in the newborn state and also indicate the importance of oxysterol 7-hydroxylase (cyp7b) activity in bile acid formation (and relief of consequences of cyp7a absence) after its induction in ~1-mo-old mice. Further studies in 3-mo-old male cyp7a / mice (944) showed a reduction in bile acid synthesis, a decrease in the intestinal bile acid pool, a very marked decrease in Chol absorption (which was reversed by feeding of cholic acid but not by administration of hyodeoxycholic acid), and increased sterol synthesis in liver and small intestine (as measured by incorporation of ³H into DPS 1 h after intraperitoneal injection of ³H₂O).

Gene expression for cyp7a has been reported to be related to changes in plasma lipoprotein Chol levels. However, the associations appear to vary in different animals and their derived genetic variants. The selectively bred hypercholesterolemic RICO rat, with elevated levels of HDL Chol in serum, showed reduced levels of hepatic microsomal cyp7a activity (684). However, reductions in the fecal excretion of bile acids or Chol were not observed. An elevated level of cyp7a expression (814) and increased fecal bile acid excretion (744) has been reported for a genetic variant of a NZW rabbit that is resistant to the effect of dietary Chol on the level of blood Chol. In short-term (3 days) experiments, hamsters infected with a cyp7a adenoviral construct showed 10- to 15-fold higher levels of hepatic cyp7a mRNA than hamsters infected with a control virus (1022). Plasma levels of LDL Chol decreased 60%. HDL Chol levels also decreased but to a lesser extent, and VLDL Chol levels were unchanged. Very recent studies in inbred strains of female mice have indicated a coordinate regulation of cyp7a gene expression in liver and the levels of HDL Chol in plasma (599). Three of the five loci for cyp7a mRNA levels on chromosomes 3, 5, and 11 were reported to be coincident with the loci for HDL Chol levels on an atherogenic diet containing Chol (1.25%) and cholic acid (0.5%). This coincidence was observed only when the animals were on the atherogenic diet. That the observed coincidence on the atherogenic diet was due to chance was considered to be remote, since the loci for the cyp7 and the loci for HDL Chol "covered only a few percent or less of the mouse genome." In another recent study, a correlation was observed between the levels, in liver, of cyp7a mRNA and apoA-I mRNA in female mice of two strains (265). In C57BL/6 mice, a direct relationship was observed between the levels of cyp7a mRNA in liver and the levels of HDL Chol in serum. This relationship was not observed in BALB/c mice, an atherosclerosis-resistant mouse strain. Other recent studies indicated genetic linkage between cyp7a and high plasma LDL Chol levels in humans (1179) and genetic relationships between alleles for cyp7a and plasma total Chol levels in the pig (817).

Aringer and Eneroth (20) reported important early studies of the conversion of labeled Chol and -sitosterol to the corresponding 5,6-epoxides, the corresponding 3,5,6-triols, and to material with the chromatographic behavior of the corresponding 7-hydroxysterols and 7-hydroxy plus 7-ketosterols upon incubation with subcellular fractions of rat liver. Their study, as well as a prior study by Danielsson (233), indicated the complexities involved in these matters and the existence of significant nonenzymatic formation of the 5,6-epoxides and the 7-oxygenated sterols in such investigations. Aringer and Eneroth (20) also reported important early studies of the enzymatic conversion of the 5,6- and 5,6-epoxides of Chol to 5,6-diOH-Chol by subcellular fractions of rat liver. Essentially no activity was observed with the 100,000-g supernatant fraction of liver. The 7-oxygenated sterols, 7-OH-Chol and 7-keto-Chol, were reported to inhibit the 5,6-epoxysterol hydrolase activity in the 18,000-g supernatant fraction of rat liver using a mixture of the 5,6- plus 5,6-epoxides of Chol (or of sitosterol). They also reported that the 5,6- and 5,6-epoxides of Chol, but not 5,6-diOH-Chol, inhibited the conversion of labeled Chol to 7-OH-Chol by the 18,000-g supernatant fraction of liver homogenates.

Watabe et al. (1186) reported the conversion of both the 5,6-epoxide and 5,6-epoxide of Chol, pregnenolone, 3-hydroxypregn-5-ene, and 3-deoxyChol to the corresponding 5,6-glycols upon incubation of the epoxides with washed bovine liver microsomes. The 5,6-glycols were characterized by GC-MS. The conversions of the 5,6- and 5,6-epoxides of Chol to the 5,6-glycol were inhibited by the aziridines 5,6-imino-5-cholestan-3-ol (1 mM) and 5,6-imino-5-cholestan-3-ol (1 mM) but they were stimulated by 3,3,3-trichloro-1-propene oxide (1 mM), commonly used as an inhibitor of the enzymatic hydrolysis of epoxides of xenobiotics. The imino derivatives of Chol (1 mM) had no effect on the hydrolysis of styrene oxide and safrole oxide, whereas the trichloro-propene oxide resulted in a complete inhibition of the hydrolysis of styrene oxide and safrole oxide. Nashed et al. (696) showed the presence of Chol 5,6-epoxide hydrolase activity in microsomes obtained from livers of rats, hamsters, mice, rabbits, and humans. The 5,6-epoxide was a better substrate than the 5,6-epoxide. With the rat liver microsomes, 5,6-diOH-Chol was reported to be "virtually the exclusive product." 5,6-Iminocholestanol has been reported to be a potent competitive inhibitor of 5,6-epoxy-Chol hydrolase in liver microsomes of the rat (696) and mouse (1188). The 5,6-epoxide of 7-dehydrocholesterol (5,6-epoxy-cholest-7-en-3-ol) also inhibits the enzyme activity in rat liver microsomes (697), and it was suggested that this inhibitor acts by covalent modification of the active site of the enzyme.

Watabe et al. (1186, 1187) reported detailed studies of the formation of 5,6-epoxides of several ⁵-steroids and their subsequent conversion to the corresponding 5,6-glycols in a system containing bovine liver microsomes, an NADPH-generating system, ferrous ion, and ADP. Incubations were carried out for 40 min at 37°C (after a 5-min preincubation of the steroids with microsomes in buffer alone) at a level of 2.82 mM (in the case of Chol, 9.26 mg/8.5 ml of final incubation mixture). Under these conditions, the conversion of ¹⁴C-labeled Chol to labeled 5,6-epoxy-Chol, 5,6-epoxy-Chol, and 5,6-diOH-Chol was reported. From the data supplied, the calculated extents of formation of the 5,6-epoxide, the 5,6-epoxide, and the 5,6-glycol were 0.38, 1.69, and 0.25%, respectively. It was reported (1187) that "the microsomal formation of 5,6-oxygenated steroids from all of the ⁵-steroids examined was negligible either when boiled microsomes were used or when ferrous ion was omitted or scavenged with EDTA (1 mM), indicating the double bond oxidation processes to be enzymatic and dependent on ferrous ion." Other steroidal substrates studied under the same conditions were 3-deoxyChol (cholest-5-ene), pregnenolone, and 3-hydroxypregn-5-ene. In each case, the -epoxide was reported as the major product, a finding similar to that reported earlier by Aringer and Eneroth (20) with Chol and -sitosterol. Characterization of the products was based on HPLC and GC-MS of the free sterols. In evaluation of this work, the very high concentration (~2.8 mM) of the sterols in the aqueous incubation mixtures is noteworthy as are the relatively low extents of metabolism. The highest extent of conversion was observed with the 3-deoxyChol (5,6-epoxide, 0.88%; 5,6-epoxide, 3.27%; and 5,6-glycol, 0.42%) which could reasonably be expected to have minimal solubility in the incubation medium. The conversion of [4-¹⁴C]Chol to the 5,6-epoxide, the 5,6-epoxide, and 5,6-diOH-Chol with bovine liver microsomes was suppressed by the use of boiled microsomes, the omission of Fe²⁺ and ADP, and by the addition of EDTA (1 mM) but was unaffected by CO. The combined data were interpreted as indicating an enzymatic peroxidative epoxidation of the ⁵-steroids not involving a cytochrome P-450 system.

Relatively little is known about the enzyme that catalyzes the formation of 5,6-diOH-Chol from either 5,6-epoxy-Chol or 5,6-epoxy-Chol. Only very slight enrichment of the enzyme from rat liver has been achieved despite apparently substantial effort (683, 1189). Nonetheless, the results of a number of varied studies indicate that hepatic microsomal Chol epoxide hydrolase is clearly distinct from much more extensively studied epoxide hydrolases of liver that act on xenobiotics and other substrates (561, 683, 697, 723, 1188, 1189).

Sevanian et al. (953) reported the presence of 5,6-epoxy-Chol in rat lung tissue. The reported level of the epoxide was extraordinarily high, i.e., 6.58 ± 0.66 mg/lung (with Chol reported at a level of 3.95 ± 0.20 mg/lung). The reported level of the epoxide may represent a typographical error, since an abstract from the same group (957) in the same year noted a level of 5.0 µg/g lung tissue. The epoxide was considered to be mostly the 5,6-isomer. Although the mechanism of formation of the 5,6-epoxysterol in lung was not studied, its high levels in lung tissue were ascribed to autoxidation of Chol. Lung tissue apparently contains 5,6-epoxy-Chol hydrolase activity (953, 958), but at a lower level than in liver (958). In contrast to more extensive studies with liver (see above), the enzyme catalyzing the hydration of 9,10-epoxystearate in lung was considered to be the same as that catalyzing the hydration of the sterol epoxide (958), and 5,6-epoxy-Chol was reported to competitively inhibit the hydration of 9,10-epoxystearate.

5,6-Epoxy-Chol, 5,6-epoxy-Chol, 5,6-diOH-Chol, and 5,6-diOH-Chol have been reported to be formed upon oxidation of LDL in the presence of CuSO₄ or upon in vitro oxidation of LDL with soybean lipoxygenase (124). Identification of the novel 3,5,6-triol was based on GC-MS comparisons with a synthetic sample (for which the detailed reaction conditions and characterization of the product were not presented). The mechanism involved in the formation of the 3,5,6-triol was not established.

Heinecke et al. (380) reported that incubation of Chol, incorporated into multilamellar vesicles containing phosphatidylcholine, with myeloperoxidase gave a mixture of products that included 5,6-epoxy-Chol, 5,6-epoxy-Chol, 6-chloro-5-cholestane-3,5-diol, and another sterol chlorohydrin of undetermined structure. In the absence of the enzyme, hypochlorous acid was reported to give the same distribution of products. Identifications of the products were based on TLC and the results of GC-MS studies of TMS and heptafluorobutyrate derivatives. The results of a previous study (1155) indicated that, whereas incubation of phospholipid-Chol vesicles with hypochlorous acid or with myeloperoxidase (in the presence of hydrogen peroxide and phosphate-buffered saline) gave chlorohydrins of the fatty acids of the phospholipid, no formation of chlorohydrins of Chol was observed. Momynaliev et al. (664) studied the action of hypochlorite on Chol in liposomes. Although the formation of chlorinated sterols was not studied, a large number of oxygenated sterols were formed. These compounds, as studied by normal-phase HPLC and GC-MS, included cholesta-3,5-dien-7-one, cholest-4-ene-3,6-diol,7-OH-Chol, 7-OH-Chol, 7-keto-Chol, 5,6-epoxy-Chol, 5,6-diOH-Chol, 3-hydroxy-5-cholestan-6-one, and 5-cholestane-3,6-dione. Carr et al. (162) reported that treatment of Chol-containing liposomes with HOCl or with myeloperoxidase in the presence of hydrogen peroxide and chloride ion, resulted in the formation of material with the TLC behavior of the chlorohydrins 6-chloro-5-cholestane-3,5-diol and 5-chloro-5-cholestane-3,6-diol (more polar than Chol on TLC) and two other less polar components, one of which appeared to be a chlorohydrin. Similar products were observed upon incubation of HOCl with red cell lipids, red cell membranes, intact red blood cells, neutrophils, and a breast carcinoma cell line (MCF7). Studies of the chlorohydrins by GC-MS or LC-MS were compromised by the instability of the chlorohydrins, with the resulting formation of products with the properties of 5,6-epoxysterols. In a subsequent study by the same group, three halohydrins of Chol were formed upon incubation of Chol-lecithin (dipalmitoyl phosphatidylcholine) (1:1) with hypochlorous acid (163). The three halohydrins were 6-chloro-5-cholestane-3,5-diol, 5-chloro-5-cholestane-3,6-diol, and 6-chloro-5-cholestane-3,5-diol. The latter compound was reported to be the major product formed upon treatment of the Chol-lecithin liposomes with HOCl or with the myeloperoxidase-H₂O₂-Cl system. The structure of the compound was established by NMR. Complete ¹³C assignments were provided as well as those for the other two halohydrins of Chol. The structures of the three chlorohydrins are shown in Figure 2.

View larger version (8K): [in this window] [in a new window]   Fig. 2. Structures of three halohydrins of cholesterol (Chol) that were formed upon incubation of Chol-lecithin (dipalmitoyl phosphatidylcholine) (1:1) with hypochlorous acid.

Hazen et al. (378) reported on the in vitro oxidation of LDL Chol with myeloperoxidase. Products, characterized by TLC and MS, included 6-chloro-5-cholestane-3,5-diol, 5-chloro-5-cholestane-3,6-diol, and another chlorohydrin for which the stereochemical orientations of the chlorine and hydroxyl functions were not established. In addition, a nonpolar (on TLC) dichlorosterol was also isolated. The orientation of the chlorine atoms in the dichlorosterol was not established. Carr et al. (162) presented a nice discussion of the question of the possible in vivo physiological significance of reactions involving HOCl leading to halogenated sterols. They noted that competing reactions with free amino groups in proteins and phospholipids (to give chloramine derivatives), with SH groups in proteins and glutathione, and with unsaturated fatty acids in various lipids have to be taken into account in considerations of this matter. In contrast, Hazen et al. (378) suggested that the action of myeloperoxidase may be important in the oxidation-halogenation of LDL Chol in the artery wall, and they further suggested that these modifications of Chol occur in acidic cell compartments. Their results indicated that, at acidic pH and at concentrations of Cl found in plasma, the formation of chlorinated derivatives of Chol proceeds in high yield. It is also important to note that myeloperoxidase has been reported to be present in atherosclerotic lesions and absent in normal arteries (241). Heinecke et al. (380) suggested that the chlorohydrins might prove to be useful "as markers for lipoproteins damaged by activated phagocytes." However, no information is available on the occurrence of the chlorohydrins in LDL modified by cellular oxidation or in atherosclerotic lesions. The in vitro effects of hypochlorite, generated by myeloperoxidase, on HDL₃ have been shown to result in predominant modification of apoA-1 and to a lesser extent modification of the unsaturated fatty acids in HDL₃ (762). Products resulting from action on Chol were not identified. Treatment of HDL₃ with NaOCl reduced its capability to accelerate Chol efflux from mouse peritoneal macrophages.

The enzymatic formation of 24,25-epoxysterols has been shown to occur via cyclization of 2,3:22,23-diepoxysqualene (910). The results of subsequent studies have indicated that a partially purified squalene epoxidase from pig liver preferentially catalyzes the conversion of squalene to 2,3-epoxysqualene relative to the conversion of 2,3-epoxysqualene to 2,3:22,23-diepoxysqualene (45). In contrast, rat liver microsomes and a partially purified enzyme from pig liver have been reported to preferentially catalyze the conversion of 2,3(S):22(S),23-diepoxysqualene to 24,25-epoxylanosterol [relative to the conversion of 2,3(S)-epoxysqualene to lanosterol] (110). A key discovery in this area was the demonstration that incubation of the 10,000-g supernatant fraction of rat liver homogenate with [2-¹⁴C]mevalonate in the presence of 4,4,10-trimethyl-trans-decalin-3,7-diol inhibited Chol formation and led to the accumulation of 2,3-epoxysqualene and 2,3:22,23-diepoxysqualene (699) (as judged by TLC). Similar findings were also made upon incubation of CHO cells with labeled acetate in the presence of the decalin derivative (699). Subsequent studies (760) with the same compound inhibited the incorporation of [³H]acetate into Chol by Hep G2 cells and showed accumulation not only of labeled 2,3-epoxysqualene and 2,3:22,23-diepoxysqualene but also (24S)-24,25-epoxy-Chol. Sexton et al. (959) reported the formation of labeled materials with the TLC behavior of 2,3-epoxysqualene, 2,3:22,23-diepoxysqualene, and "polar compounds" (believed to be polar sterols) upon incubation of [³H]acetate with rat intestinal epithelial cells in the presence of 3-[2-(diethylamino)ethoxy]androst-5-en-17-one (U-18666A). No definitive characterization of these labeled materials was presented.

Nelson et al. (701) reported the formation of (24S)-24,25-epoxy-Chol upon aerobic incubation of ³H-labeled 2,3:22,23-diepoxysqualene with a rat liver homogenate preparation. No conversion of either squalene, 2,3(S):22(S),23-diepoxysqualene, or (24S)-24,25-epoxy-Chol to 25-OH-Chol was detected upon their incubation with rat liver homogenate preparations. Nelson et al. (700) reported that small amounts of material with the properties of 24,25-epoxy-Chol are formed upon incubation of rat liver homogenate preparations with [¹⁴C]acetate in the absence of an inhibitor of 2,3-epoxysqualene cyclase. Steckbeck et al. (1039) reported that labeled (24R)-24,25-epoxy-Chol was not detected upon incubation of a rat liver homogenate preparation with [2-³H]-(24R),25-epoxylanosterol. However, labeled materials with the properties of (24R)-24-hydroxylanost-8-en-3-ol and (24R)-24-OH-Chol were reported. Taylor et al. (1102) reported the conversion of (24S),25-epoxylanosterol to (24S)-24,25-epoxy-Chol in both mouse L cells and Chinese hamster lung cells. The same workers reported that (24R),25-epoxylanosterol was converted to (24R)-24,25-epoxy-Chol in mouse L cells and, in Chinese hamster lung cells, to (24R)-24-OH-Chol. Saucier et al. (897) reported the formation of (24S)-24,25-epoxy-Chol from labeled mevalonate in CHO cells. Favata et al. (293) observed that incubation of [¹⁴C]acetate with CHO cells or a CHO mutant defective in the 14-demethylation of lanosterol in the presence of ketoconazole, at concentrations greater than 5.0 µM, led to the formation of material with chromatographic behavior (reverse-phase HPLC) of 2,3:22,23-diepoxysqualene and 24,25-epoxylanosterol. Panini et al. (757) reported the formation of ¹⁴C-labeled 24,25-epoxylanosterol upon incubation of CHO-K1 cells with [1-¹⁴C]acetate in the presence of ketoconazole (15 µM). Full characterization of the labeled product was not presented. Panini et al. (758) reported the formation of labeled (24S)-24,25-epoxylanosterol after incubation of rat intestinal epithelial (IEC-6) cells with [³H]acetate in the presence of U-18666A (to cause an accumulation of 2,3:22,23-diepoxysqualene) followed by further incubation of the cells with fresh medium containing ketoconazole (an inhibitor of sterol 14-demethylation). The product was characterized by chromatography. Panini et al. (759) reported that incubations of rat intestinal epithelial (IEC-6) cells with [³H]acetate in the presence of progesterone (10 µg/ml) caused an accumulation of materials with the chromatographic behavior (reverse-phase HPLC) of desmosterol (cholesta-5,24-dien-3-ol) and (24S)-24,25-epoxy-Chol.

Dollis and Schuber (259) found that incubation of Hep G2 cells with [2-¹⁴C]acetate in the presence of a substituted azadecalin (N-[1,5,9-trimethyldecyl]-4,10-dimethyl-8-aza-trans-decal-3-ol), an inhibitor of 2,3-epoxysqualene-lanosterol cyclase, resulted in the accumulation of 2,3-epoxysqualene, 2,3:22,23-diepoxysqualene, and 24,25-epoxy-Chol (characterized by TLC). The same workers also reported the conversion, by Hep G2 cells, of added 2,3:22,23-diepoxysqualene to 24,25-epoxy-Chol (characterized by GC and GC-MS). Mark et al. (617) described the chemical synthesis of a new inhibitor (BIBX 79) of 2,3-epoxysqualene formation. The potency of this compound was reported to be considerably higher than that reported previously for inhibitors of this enzyme. The IC₅₀ value for inhibition of the incorporation of [2-¹⁴C]acetate into DPS by Hep G2 cells was reported to be 3.8 × 10⁹ M. The IC₅₀ value for the conversion of ¹⁴C-2,3-epoxysqualene to lanosterol or epoxylanosterol by the Hep G2 cells was very similar (6 × 10⁹ M). BIBX 79 was reported to cause the accumulation of significant amounts of 2,3-[¹⁴C]epoxysqualene and diepoxysqualene after incubation of Hep G2 cells with labeled acetate. Lesser amounts of ¹⁴C were observed to be associated with "epoxycholesterol" and "desmosterol." These results were based on reverse-phase HPLC. This chromatographic system almost certainly is incapable of separating various C₂₇ sterols from each other (869) and provided only slight separation of standards of Chol and lanosterol. Thus a number of studies indicate the formation of 24,25-epoxylanosterol (and of 24,25-epoxy-Chol formed from it) via enzymatic cyclization of 2,3:22,23-diepoxysqualene. 24,25-Epoxysterols could also arise from direct enzymatic epoxidation of ²⁴-sterols, although this process has not, to my knowledge, been described in mammalian systems. 24,25-Epoxysterols could also result from autoxidation of ²⁴-sterols or via epoxidation secondary to peroxidation of other lipids.

Tabacik et al. (1083) reported the formation of labeled "lanost-3-ol-32-al" by human lymphocytes incubated with sodium [2-¹⁴C]mevalonate. The evidence for the structural assignment, while suggestive, was limited. A cytochrome P-450 involved in the removal of the 14-methyl group of sterol precursor of Chol has been purified to varying extents from rat liver microsomes (947, 1124) and pig liver microsomes (1016). Trzaskos and co-workers (1126, 1127) reported incubation conditions favoring the accumulation of lanost-8-ene-3,32-diol and 3-hydroxylanost-8-en-32-al (not resolved in the reverse-phase HPLC system used, Ref. 1126) upon incubation of [24,25-³H]lanost-8-en-3-ol with rat liver microsomes. These included limiting levels of NADPH in the presence of NADH (1126), elevated pH (1127), limiting enzyme protein levels (1127), and incubation in the presence of miconazole or ketoconazole (1127). Saucier et al. (898) presented evidence indicating the formation of 32-oxolanosterol and 32-hydroxylanosterol in Chinese hamster lung cells incubated in the presence of a very high level (23 mM) of sodium mevalonate. Although neither sterol was detected in control cells, the levels of the 32-oxo and 32-hydroxysterols in the treated cells were reported to be, in two experiments, 0.058 and 0.084 µg/mg protein for the 32-oxosterol and 0.0088 and 0.0120 µg/mg protein for the 32-hydroxymethylsterol. Structure assignments were based on HPLC and limited mass spectral and ¹H-NMR data along with chromatographic studies of the product of borohydride reduction of the putative 32-oxo sterol.

A number of inhibitors of the 14-demethylase have been described. Included are ketoconazole (361, 1016, 1127, 1133), miconazole (430, 734, 1127), 14-ethyl-5-cholest-7-ene-3,15-diol (430, 679, 805, 806), 14-methyl-5-cholest-7-ene-3,15-diol (679), lanost-7-ene-3,15-diol (679), 3-hydroxylanost-8-en-7-one (17, 947), 14-alkyl-substituted lanosterol analogs (1132, 1133), and 3-hydroxylanosta-8,15-diene-32-carboxylic acid (627). The latter synthetic compound was reported to be highly active in the inhibition of 14-demethylase activity in Hep G2 cells, with an IC₅₀ value of 2 nM (627).

The cloning of the cDNA encoding human and rat liver 14-demethylase has been reported (16, 868, 996, 1054). Approximately 93% homology in the deduced amino acid sequences for the rat and human enzymes was observed (16, 1054). Approximately 38-42% homology was observed between the rat sequence and those reported previously for the protein from fungal sources. Strömstedt et al. (1054) reported that the human 14-demethylase gene was expressed in a variety of tissues with the highest levels observed in testes, ovary, adrenal, prostate, liver, kidney, and lung. Transfer of either human adrenocortical (H295R) cells or human hepatoma (Hep G2) cells from medium containing 10% bovine serum to medium containing 10% delipidated bovine serum resulted in increased levels of mRNA for the 14-demethylase, suggesting regulation by Chol (or other lipid constituents of serum). The increase was more substantial in the adrenal cells. 25-OH-Chol (12.4 µM) resulted in substantial decreases in the levels of mRNA for the 14-demethylase. Thus transcription of the 14-demethylase gene may be regulated by other oxysterols. No other oxygenated sterols were studied in this regard.

The levels of the enzyme catalyzing the oxidation of the 14-methyl group of Chol precursors appear to be under physiological regulation. Chol feeding (3% in diet for 1 or 4 wk) to male Wistar rats (150-160 g) lowered the level of lanosterol 14-demethylase (cyp51) activity and cyp51 content in liver microsomes (1017). After 1 wk of Chol feeding, 14-demethylase activity and P-450_14DM content were reduced 38 and 33%, respectively. After 4 wk of Chol feeding, cyp51 activity and cyp51 protein were reduced 45 and 44%, respectively. Chol feeding (1 and 4 wk) had no effect on the levels of microsomal NADPH-P-450 reductase activity or total cytochrome P-450. The level of activity of cyp51 was increased in ovary (but not liver) after subcutaneous administration of gonadotrophin (pregnant mare's serum) (1219). Germ cells from mature rats showed higher cyp51 activity than in those from prepubertal animals (1055).

The human 14-demethylase gene has been localized to chromosome 7 (7q21.2-q21.3) and has been shown to contain 10 exons (867). The mRNA levels for the 14-demethylase (ERG11) of the yeast Saccharomyces cerevisiae increased upon incubation with glucose or heme and increased under low oxygen growth conditions (1136). Interestingly, cytochrome P-450 reductase and the 14-demethylase of the yeast appeared to be coordinately regulated. The nucleotide sequence for lanosterol 14-methylase from Candida albicans has been reported (536).

Whereas the formation of 14-hydroxymethyl and 14-formyl sterols has been demonstrated in mammalian tissues, the occurrence of 14-carboxylic acid derivatives of sterols has not been shown in animals, with the exception of certain sponges. Cheng et al. (195) reported the occurrence of a 14-carboxylic acid derivative of lanosterol in an Okinawan sponge (Penares sp.). This new sterol, penasterol (Fig. 3A), was reported to show in vitro antitumor activity (IC₅₀ 7.9 µM) against L1210 mouse leukemia cells. Shoji et al. (977) reported the occurrence of two other 14-carboxylic acid derivatives of lanosterol in an Okinawan sponge (Fig. 3, B and C).

View larger version (8K): [in this window] [in a new window]   Fig. 3. Structures of penasterol (3-hydroxylanosta-8,24-dien-32-oic acid) (A), 3-oxo-8,24-dien-32-oic acid (B), and 3-acetoxylanosta-8,24-dien-32-oic acid (C).

Sonoda et al. (1018) described the chemical synthesis of the ⁷-analog of penasterol, which was reported to show in vitro antitumor activity against L1210 mouse leukemia cells (IC₅₀ 2.3 µM) and human epidermoid carcinoma KB cells (IC₅₀ 7.2 µM). The methyl ester derivative showed little or no activity under the conditions studied. Other oxygenated derivatives of 24,25-dihydrolanosterol were also reported to show in vitro antitumor activity, most notably the (24R)-24,25-epoxide and the 24-keto derivatives.

It is noteworthy that several substituted ent-5,14-androstane derivatives with a 14-carboxymethyl function have been isolated from a Penicillium species and shown to have in vitro inhibitory activity against partially purified farnesyltransferase from human cells (732, 1141). The structures of three new compounds, termed andrastatins A, B, and C, are shown in Figure 4. The potencies (IC₅₀) of andrastatins A, B, and C against farnesyltransferase were 24.9, 47.1, and 13.3 µM, respectively.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Structures of andrastatins A, B, and C.

The presence of 4-OH-Chol in human blood (121, 128) and in rat liver has been reported (121). The origin of this oxysterol has not been established.

19-OH-Chol has been reported by two groups (520, 1108) to be present in membranes of erythrocytes from patients with sickle cell anemia. The reported occurrence of 19-OH-Chol in animal tissues is, to the knowledge of the reviewer, without precedent. Hydroxylation of the C-19 methyl group of C-19 steroids is known to occur in the series of reactions involved in the formation of estrogens. However, enzymatic 19-hydroxylation of C₂₇ sterols has not been described in mammalian systems. Nonetheless, it should be noted that Gustafsson et al. (366) suggested the presence of a 19-hydroxylated bile acid as a metabolite of labeled lithocholic acid by microsomes from human fetal liver. Furthermore, Kurosawa et al. (525) reported the occurrence of a 19-hydroxylated bile acid, i.e., 3,7,12,19-tetrahydroxy-5-cholan-24-oic acid in the urine samples from 2- to 9-day-old healthy human infants. This bile acid was present at 0.1-1.5 µg/ml and corresponded to an estimated 1.5 to 7.0% of total urinary bile acids. The 19-hydroxylated bile acid was characterized by GC-MS studies with comparisons with an authentic standard prepared by chemical synthesis. The 19-hydroxylated bile acid was not detectable in the urine of older healthy children or adults. In an earlier study, Gustafsson et al. (366) reported evidence indicating 19-hydroxylation of lithocholic acid by microsomes of fetal human liver. [24-¹⁴C]3-hydroxy-5-cholan-24-oic acid (lithocholic acid) was incubated with liver microsomes of fetal (14-24 wk) human subjects in the presence of NADPH. The products were subjected to TLC, and one radioactive zone was found to contain material which, as its TMS derivative, showed a MS that was compatible with the TMS derivative of a primary alcohol. The structure 3,19-dihydroxy-5-cholan-24-oic acid was suggested. An authentic sample was not available for comparison. Kimura et al. (491) reported low levels of the 19-hydroxy derivative of cholic acid in the urine of newborn infants. Even lower levels were reported for urine obtained from women in late pregnancy and shortly after delivery. Identification of the 19-hydroxylated bile acid was based on GC-MS of the dimethylethylsilyl ether derivative of the methyl ester. Full MS data were not presented.

3-Hydroxy-5-cholest-8(14)-en-15-one has been reported to be present in rat skin and rat hair (278). The mode of formation of the 15-ketosterol is not known. However, in view of the demonstration of the occurrence of 5-cholest-8(14)-en-3-ol in rat skin (551, 597), it is possible that the 15-ketosterol in rat skin and hair might have arisen from autoxidation of the ⁸⁽¹⁴⁾-sterol present in skin (and presumably in hair). The results of a control experiment, including the addition of ⁸⁽¹⁴⁾-sterol to hair before processing of the sample, demonstrated that the 15-ketosterol isolated was not formed by autoxidation of the ⁸⁽¹⁴⁾-sterol during the procedures utilized in the purification and analysis of the 15-ketosterol. However, the possibility exists that some or all of the 15-ketosterol found to be present in rat skin and hair was formed by autoxidation of the ⁸⁽¹⁴⁾-sterol before the death of the rats. It is possible that 5-cholest-8(14)-en-3-ol served as a precursor of the 15-ketosterol, either by enzymatic action or by autoxidation.

The development of conditions for large-scale incubations of 2,3-epoxysqualene with baker's yeast (144) provided the opportunity to explore this system for the preparation of various analogs of lanosterol using appropriately substituted 2,3-epoxysqualenes as substrates. With the use of the synthetic methyl ester as substrate, the methyl ester of ganoderic acid Z was obtained (Fig. 5).

View larger version (7K): [in this window] [in a new window]   Fig. 5. Synthesis of ganoderic acid Z methyl ester by cyclization of a derivative of 2,3-epoxysqualene with baker's yeast.

The same approach was utilized for the preparation of the 4-hydroxymethyl analog of lanosterol (634). Exploiting this approach further, Medina et al. (633) provided a novel method for the preparation of C-32 oxygenated sterols. They observed the cyclization of the appropriately vinyl-substituted analog of 2,3-epoxysqualene to give the 14-vinyl analog of lanosterol (Fig. 6).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Synthesis of 32-oxygenated derivatives of lanosterol via cyclization of a vinyl-substituted analog of 2,3-epoxysqualene with baker's yeast.

Xiao and Prestwich (1206) utilized the same approach to prepare 21-hydroxylanosterol and 19-hydroxylanosterol from appropriately substituted analogs of 2,3-epoxysqualene.

Chol oxidase has been used, on a preparative scale, to synthesize the 3-keto derivatives of a number of oxygenated sterols from the corresponding 3-hydroxysterols (769, 779, 924, 931, 935, 938). The use of this commercially available enzyme provided a powerful approach for the selective oxidation of the 3-hydroxy function in sterols with multifunctional substituents. Recent results of Teng and Smith (1109) indicate that careful attention to product identification cannot be bypassed since, with Chol oxidase of Pseudomonas fluorscens, oxidation of Chol gave 6-hydroperoxycholest-4-en-3-one as the major product, instead of the expected cholest-4-en-3-one. Oxidation of 25-OH-Chol under the same conditions gave material with the expected chromatographic and ¹H-NMR spectral properties of 6-hydroperoxy-25-hydroxycholest-4-en-3-one. Oxidation of 19-OH-Chol by the enzyme from P. fluorescens was reported to give four products, the identity of which was not established. In an earlier study, Liu et al. (581) reported that incubation of rabbit LDL with commercial Chol oxidase (from P. fluorescens) resulted in the conversion of Chol in LDL to 20-OH-Chol and 25-OH-Chol. Identification of the latter compounds was based solely on HPLC analysis, and these assignments of structure should be viewed with caution. A more recent study from the same laboratory (836) claimed the formation of 25-OH-Chol and 20-OH-Chol from Chol present in rat liver nuclei upon incubation with Chol oxidase. Again, the assignment of structure for the two oxysterols was based solely on HPLC. Smith and Brooks (997) studied the rates of oxidation of different side-chain oxygenated derivatives of Chol by Chol oxidase from Nocardia erythropolis. Varying maximum velocity values (relative to Chol as 1.00) were reported as follows: (20S)-20-OH-Chol, 1.04; (22RS)-22-OH-Chol, 0.90; (20R,22R)-20,22-diOH-Chol, 0.93; 24-OH-Chol, 1.29; 24-keto-Chol, 1.32; 25-OH-Chol, 0.61; and (25R)-26-OH-Chol, 0.56.

Marco de la Calle and co-workers (614, 615) studied the incorporation of tritium into hepatic sterols of liver of male Wistar rats at 1 h after the intraperitoneal injection of ³H₂O. Significant incorporation of the labeled hydrogen into "polar sterols" was reported. Chol feeding (1% in diet for 24 h) was reported to decrease HMG-CoA reductase activity in liver, to decrease the incorporation of the labeled hydrogen into NSL and Chol, and to increase the percentage of ³H of NSL that was associated with polar sterols. In the Chol-fed animals, strong inverse relationships were reported (615) between the accumulation of labeled polar sterols and the levels of HMG-CoA reductase activity, and the incorporation of labeled hydrogen into NSL and "Chol" (characterized only by TLC). In other experiments, intragastric administration of ketoconazole (24 mg/kg) was reported to cause, at early time points (2-12 h), a decrease in hepatic HMG-CoA reductase, a decreased in vivo incorporation of ³H₂O into Chol, and an increase in the percentage of ³H of NSL that was associated with polar sterols (679). The authors ascribed the polar sterols to oxysterols and suggested that Chol feeding (or ketoconazole treatment) resulted in the accumulation of oxygenated Chol precursors. They also noted that such precursors (e.g., 32-hydroxylanosterol and the corresponding 32-aldehyde), known to decrease HMG-CoA reductase activity in cultured cells, might be the polar sterols responsible for the lowering of HMG-CoA reductase activity in the livers of the Chol-fed (or ketoconazole-treated) animals. A very important limitation of this work lies in the lack of identification of the labeled polar sterols or even the establishment that the concerned materials were, in fact, sterols. Definition of this labeled material was limited to TLC mobility, i.e., "the region of the plate between the origin and Chol." Nonetheless, the interesting findings reported should provide the impetus for reinvestigation of this matter with more rigorous methodologies.

Breuer and Björkhem (123) reported very interesting results relative to the origin of certain oxysterols in plasma and in liver. In one experiment, one rat was exposed to ¹⁸O₂ for 178 min, after that ¹⁶O₂ was introduced for 25 min. Blood samples were taken at 0, 178, and 205 min. The livers were homogenized in phosphate buffer containing butylated hydroxytoluene (BHT). The plasma and liver homogenate were frozen and stored for 20 days at 20°C. One control rat, exposed to ¹⁶O₂, was studied similarly. Oxysterols were analyzed after mild alkaline hydrolysis and workup as described previously by the authors (273) except that TBDMSi ethers were analyzed by GC-MS. The ¹⁸O content of various oxysterols, Chol, and 5-cholest-7-en-3-ol was reported. With the exception of some of the studies with liver in one experiment, it appears that all of the results reflect a single analysis of samples from one animal. No data on the levels of the various sterols were provided. This may be relevant to some observations made since two sterols of particular interest with regard to ¹⁸O incorporation, i.e., 7-OH-Chol and 25-OH-Chol, were reported in another study from the same laboratory (273) to be present in human plasma at very low levels, i.e., 0.008 ± 0.12 and 0.005 ± 0.007 µM, respectively. The authors noted that with the design used in the first experiment "there is a theoretical risk that ¹⁸O₂ dissolved in plasma or bound to hemoglobin might oxidize cholesterol during sample processing." To deal with this, a second experiment was carried out in which the animal was exposed to normal air for 5 min after the period of exposure to an atmosphere containing ¹⁸O₂. "After 183 min of ¹⁸O₂ exposure, ¹⁶O₂ was delivered to the cage for 22 min. The cage was then opened wide at 210 min to allow equilibration with room air for 5 min." Blood samples were taken at 0 and 210 min. The liver was also removed at 210 min. In both experiments, very little or none of the Chol in plasma or liver contained ¹⁸O. In the first experiment, plasma Chol contained 1% ¹⁸O species at 178 and 205 min and 2% ¹⁸O species in Chol of liver at 205 min. In the second experiment, plasma and liver Chol contained 3 and 5% of ¹⁸O species at 210 min, respectively. In contrast, 5-cholest-7-en-3-ol contained substantial amounts of ¹⁸O in both experiments. In the first experiment, the plasma 5-cholest-7-en-3-ol contained 52 and 54% of ¹⁸O species at 178 and 205 min, respectively, and the liver 5-cholest-7-en-3-ol contained 61% of ¹⁸O species at 205 min. Very similar results were obtained in the second experiment. The plasma and liver 5-cholest-7-en-3-ol contained 60 and 56% ¹⁸O species at 210 min, respectively. The difference between the results for the ⁷-sterol and Chol is undoubtedly related to the marked difference in the steady-state concentrations of the two sterols in liver and plasma and that a substantial fraction of the 5-cholest-7-en-3-ol represented newly synthesized sterol that was not substantially diluted by a large pool of the endogenous ⁷-sterol.

The results with the oxysterols are less amenable to simple interpretation. The authors concluded that "in vivo formation of oxysterols, indicated by enrichment in ¹⁸O, was established for cholest-5-ene-3,7-diol, cholest-5-ene-3,7-diol, 7-oxocholesterol, cholest-5-ene-3,24-diol, cholest-5-ene-3,25-diol, and cholest-5-ene-3,27-diol. Additionally, it seems likely that cholest-5-ene-3,4-diol is formed in vivo. The ¹⁸O labeling pattern suggests that there is incomplete equilibration between liver and plasma pools of cholest-5-ene-3,27-diol. No evidence for the in vivo formation of 5,6-oxygenated oxysterols was obtained." Because these results have been cited and discussed in later publications (121, 591), a close inspection of these results is warranted. Despite the conclusions made by the authors, the interpretation of the observed data appears to be less than simple. Substantial ¹⁸O incorporation was observed with most, but not all, of the oxysterols studied.

Two of the oxysterols studied, 26-OH-Chol and 24-OH-Chol, are known as products of P-450-dependent hydroxylation of Chol and are not considered to be significant autoxidation products of Chol. Interestingly, the ¹⁸O content of these two sterols differed markedly. In both experiments, the presence of two atoms of ¹⁸O was not detected in 24-OH-Chol from plasma. ¹⁸O was detected in 24-OH-Chol from plasma but only as a mono-¹⁸O species. The levels of mono-¹⁸O species in plasma were 17% at 178 min and 11% at 205 min in the first experiment and 9% at 210 min in the second experiment. No detectable ¹⁸O was found in 24-OH-Chol in liver in either the first or second experiments. In a subsequent report from the same laboratory (596), the level of mono-¹⁸O species in brain at 210 min in the second experiment was reported as 11%. No mention of species containing two atoms of ¹⁸O was made. In another subsequent report from the same laboratory (94), the much lower ¹⁸O content of 24-OH-Chol (relative to 26-OH-Chol and 7-OH-Chol) in plasma was ascribed to the lower rate of synthesis of the 24-OH-Chol in relation to its pool size. In contrast to these results with 24-OH-Chol, 26-OH-Chol in plasma was substantially enriched with ¹⁸O species, most of which were present as mono-¹⁸O species (in both experiments). For example, in the first experiment, 26-OH-Chol of plasma contained 47 and 8% as mono- and di-¹⁸O species at 178 min, respectively, and 50 and 4% as mono- and di-¹⁸O species at 205 min, respectively. In the second experiment, 26-OH-Chol from plasma at 210 min contained 47 and 4% as mono- and di-¹⁸O species, respectively. The ¹⁸O content of 26-OH-Chol in plasma was substantially higher than that of 26-OH-Chol in liver. This difference was ascribed by the authors to "an incomplete equilibration between the liver and plasma pools" of this sterol.

4-OH-Chol has been reported to occur in human plasma (121, 128). However, its origin (i.e., enzymatic or via autoxidation) has not been established. The ¹⁸O content of 4-OH-Chol in plasma and liver was relatively low, and all of the ¹⁸O appeared to be mono-¹⁸O species. No ¹⁸O enrichment was observed with either 5,6-epoxy-Chol or 5,6-diOH-Chol. 25-OH-Chol and 7-OH-Chol can arise from both enzymatic and autoxidation of Chol. The 25-OH-Chol and 7-OH-Chol in plasma contained mostly mono-¹⁸O species (in both experiments). The same was reported for these two sterols from liver. In the first experiment, the 7-OH-Chol in plasma contained 21 and 19% mono-¹⁸O species at 178 and 205 min, respectively, and 41% mono-¹⁸O species at 210 min in the second experiment. The 25-OH-Chol in plasma contained 57 and 50% mono-¹⁸O species at 178 and 205 min, respectively, in the first experiment and 50% mono-¹⁸O species at 210 min in the second experiment. 7-OH-Chol in liver contained 11 and 25% mono-¹⁸O species in the first and second experiments, respectively. The ¹⁸O in liver 7-OH-Chol from one experiment was reported to be mostly located at C-7. The 25-OH-Chol in liver was reported to contain 54 and 42% mono-¹⁸O species in the first and second experiments, respectively. 7-Keto-Chol is a known product of autoxidation of Chol and of lipid peroxidation. 7-Keto-Chol from the plasma and liver showed moderate levels of ¹⁸O incorporation, and all of this ¹⁸O was in mono-¹⁸O species.

In contrast to all of the oxysterols noted above, a very substantial fraction of the ¹⁸O in 7-OH-Chol was in di-¹⁸O species. This was true for samples from both plasma and liver. For example, in the second experiment, the 7-OH-Chol from plasma contained 59 and 23% of mono- and di-¹⁸O species, respectively, and the sample from liver contained 29 and 24% of mono- and di-¹⁸O species, respectively. The 7-OH-Chol from liver, when analyzed by GC-MS as its diTMS ether derivative, showed (based on M⁺) 29% mono-¹⁸O species and 22% di-¹⁸O species. Based on M-90 (with the authors' assumption that this ion represents specific loss of O at C-3), only mono-¹⁸O species (34%) were observed. The unique high incorporation of two atoms of ¹⁸O in the 7-OH-Chol prompted the following statement from the authors " ... it is tempting to suggest that cholest-5-ene-3,7-diol is predominantly formed from newly synthesized Chol, or a Chol precursor, by enzymatic reactions in vivo." It should be noted that substantial di-¹⁸O species were observed only with 7-OH-Chol. With all other oxysterols the estimated levels of di-¹⁸O species were, in almost all samples, between 2 and +4%. In view of the fact that oxidation of Chol with ¹⁸O₂ in the presence of Cu²⁺ gave ¹⁸O-labeled 7-OH-Chol with 6% di-¹⁸O species, it might be surmised that, within the apparent errors of the experiment, essentially no di-¹⁸O species were observed for the various oxysterols other than 7-OH-Chol. It seems reasonable to assume that, in these oxysterols, the ¹⁸O was not at C-3 but was at the site of the oxygen substitution. However, of these oxysterols, evidence on this point was provided only for one sample of 7-OH-Chol from the liver obtained in one experiment. Also, it is assumed that the presence of ¹⁸O in the oxysterols establishes their enzymatic formation in vivo. The second experiment was carried out in such a manner as to make it unlikely that residual ¹⁸O₂ dissolved in plasma or bound to hemoglobin could oxidize the Chol during sample preparation. However, direct evidence on this point was not provided. In addition, the possibility that significant levels of ¹⁸O might be present in a fatty acid hydroperoxide (594) (or a similar species with a potentially longer biological and chemical half-life) existed at the end of the experiment, which could provide a source of ¹⁸O for subsequent autoxidation of Chol, was not excluded. In addition, because the level of 7-OH-Chol in plasma has been reported to be very low (121, 273, 521), any ¹⁸O-labeled 7-OH-Chol would undergo very little dilution with endogenous unlabeled 7-OH-Chol (relative to other oxysterols).

Johnson et al. (456) reported on the formation of labeled oxysterols in liver 1 h after the intraperitoneal injection of [5RS-³H]mevalonolactone to one control rat and to one Chol-fed (5% in Chol-free diet) rat. The rats were fasted for 6 h before feeding for 1 h of Chol (in a Chol-free diet) or, in the case of the control rat, the Chol-free diet alone. The livers were frozen in liquid nitrogen and then saponified with treatment with a solution containing 50% aqueous KOH (2 parts) and 95% ethanol (3 parts) "at reflux temperature for 3 h in the dark in an atmosphere of nitrogen." Under these conditions, essentially quantitative decomposition of any 7-keto-Chol would be anticipated (273, 521, 603, 771). A small amount of BHT was added as an antioxidant. The NSL were recovered by extraction with hexane. After separation of polar oxysterols from Chol by reverse-phase flash chromatography on a silica gel (C₁₈ bonded-phase) column, the labeled polar sterols were subjected to normal-phase HPLC. In the liver from the control rat, two major labeled components with the reported mobility of 7-keto-Chol and 7-OH-Chol were observed. The mobilities of unlabeled standards corresponding to the labeled sterols were not shown (nor those of other oxysterols). In the liver of the rat, which received the Chol-containing meal for 1 h, three major peaks were observed that were said to correspond to 25-OH-Chol, 7-keto-Chol, and 7-OH-Chol. No further characterization of the labeled materials was made. The reported substantial formation of material with the chromatographic mobility of 7-keto-Chol is noteworthy in view of the saponification conditions employed (see above). The authors claimed that the peaks corresponding to 7-keto-Chol and 7-OH-Chol were labeled with tritium "to a greater extent" in the Chol-fed rat than in the control rat and that this was "consistent with a stimulation of bile acid formation in response to the Chol challenge." These claims must be viewed with caution in view of the lack of complete characterization of the labeled materials and the use of an experimental design involving the use of only one experimental rat and one control rat. Johnson et al. (456) also reported the incorporation of labeled hydrogen of water into several oxysterols of livers from rats fed a Chol-containing meal. The rats (number not clearly stated for this particular experiment) were given D₂O (33% in distilled water) ad libitum as drinking water for 3 days before death. Control animals received no D₂O. The animals were fasted for 6 h (5-11 A.M.). The experimental rats were fed for 1 h with a Chol-containing (5%) diet; the control group was fed the same diet containing purified Chol (5%). After an additional hour, the rats were killed, and the pooled liver samples were then analyzed for deuterium incorporation into various sterols. The incorporation of deuterium into the following oxysterols was reported: 7-OH-Chol, 26-OH-Chol, and 25-OH-Chol. The extents of incorporation of the deuterium into 7-OH-Chol and 25-OH-Chol were similar, whereas that into 26-OH-Chol was lower. It was reported that no incorporation into 7-OH-Chol was observed. No other oxysterols were mentioned. The incorporation of deuterium was studied in a special way, based only on the enrichment of the M+1 ion on GC-MS.

Lund et al. (593) reported increases in the levels of 24-OH-Chol and 26-OH-Chol in liver after administration of Chol (2% in diet for 4 days). Nonetheless, the authors concluded that "neither 24-hydroxylation nor 27-hydroxylation are critical for the cholesterol-induced downregulation of HMG-CoA reductase in mouse liver." This conclusion was based on the results of in vivo experiments in which deuterium-labeled analogs of 26-OH-Chol or 24-OH-Chol were fed to mice. The effects of feeding either Chol, [23,23,24,24,25-²H₅]Chol, or [26,26,26,27,27,27-²H₆]Chol at levels of 0.05% by weight in diet to mice on the levels of liver microsomal HMG-CoA reductase activity were studied. The Chol samples (0.05% by weight in diet) were fed in diet supplemented with peanut oil (10% by weight in diet). The mice were fed individually 6 g of food for 24 h; then "the mice were again fed individually in the morning with 6 g of the sterol-containing diet and killed 24 h later." No information was provided as to whether or not the animals were on a normal light-dark cycle or on the actual times at which the bulk of the food was consumed. The results indicated that the animals fed Chol or the deuterated Chol samples each showed mean levels of HMG-CoA reductase activity that were ~50% that of control animals (no added Chol). The authors anticipated a reduced suppression of HMG-CoA reductase activity in animals fed the 26-OH-Chol and 24-OH-Chol (if the formation of these sterols from Chol was important in the lowering of reductase activity) due to the presence of isotope effects observed in their formation from Chol in mouse liver mitochondrial incubations. The absence of an isotope effect in the lowering of HMG-CoA reductase activity in liver (i.e., the lack of a reduced suppression of reductase activity in animals receiving the deuterated Chol samples) was presented as major evidence "that 24- and 27-hydroxylation are not involved in the Chol-induced downregulation of HMG-CoA reductase activity in mouse liver." Lund et al. (593) reported an isotope effect in the formation of 26-OH-Chol from [26,26,26,27,27,27-²H₆]Chol and [25,26,26,26,27,27,27-²H₇]Chol and in the formation of 24-OH-Chol from [23,23,24,24,25-²H₅]Chol and [24,24-²H₂]Chol by mouse liver mitochondria in the presence of isocitrate and NADPH. The GC-MF analyses were made of the molecular ion region of the TMS ether derivative of the 26-OH-Chol formed from a 1:1 mixture of [6,7,7-²H₃]Chol and [26,26,26,27,27,27-²H₆]Chol or [25,26,26,26,27,27,27-²H₇]Chol during a 1-h incubation with the mouse liver mitochondria. The GC-MF analyses were also made of the ion corresponding to M-TMSOH-43 (isopropyl function) region of the MS of the TMS derivative of the 24-OH-Chol formed from a 1:1 mixture of [6,7,7-²H₃]Chol and [24,24-²H₂]Chol or [23,23,24,24,25-²H₅]Chol during a 1-h incubation with the mouse liver mitochondria. From these 1-h incubations, the authors estimated a K_H/K_D of 2.5 for the formation of the 26-hydroxysterol and a K_H/K_D of 4.5 or 4.3 for the formation of the 24-hydroxysterol. Whereas these results strongly indicate the presence of isotope effects, the estimates of the magnitude at K_H/K_D based on a single time determination of the amount of product formed from the precursor labeled with deuterium at the concerned carbon atom and its analog lacking the deuterium substitution is subject to criticism. This follows from the simple consideration that the ratios of products from the deuterated and nondeuterated precursors can be expected to vary considerably with the extent of conversion of the precursors to Chol. Moreover, in the case of the 26-OH-Chol, the extent of its further metabolism (conversion to the corresponding aldehyde and carboxylic acid) by the mitochondria under the conditions studied, as well as the presence or absence of isotope effects in the concerned reactions, should be considered. In the case of the experiments involving the in vivo feeding of the deuterated Chol samples, the experimental system should be recognized as considerably more complex. The lack of observation of isotope effects in the in vivo experiments does not, for this reviewer, justify any conclusion with regard to the involvement of 26-OH-Chol or 24-OH-Chol in the regulation of hepatic HMG-CoA reductase.

Interesting results on the side-chain oxidation of deuterium-labeled Chol by highly purified cytochrome P-450₂₆ from pig liver mitochondria were reported by Lund et al. (592). The purified hydroxylase was incubated with the Chol samples (added in acetone) in the presence of adrenodoxin, adenodoxin reductase, and NADPH for 30 min in Tris acetate buffer containing 20% glycerol and 0.1 mM EDTA. Under these conditions, the reconstituted cytochrome P-450 system was reported to catalyze the formation of 26-OH-Chol, 25-OH-Chol, and 24-OH-Chol. In contrast to the case of the studies with mouse mitochondria, no significant isotope effects were observed for the 26-hydroxylation (or the 25-hydroxylation). However, a marked (and much higher than for the mouse system) isotope effect K_H/K_D of >10 was reported for 24-hydroxylation of Chol. These results were based on analyses of sterol products after a 30-min incubation period. 26-Hydroxylation was assayed by GC-MS of the TMS derivative after incubation of a 2:1 mixture of [6,7,7-²H₃]Chol and [25,26,26,26,27,27,27-²H₇]Chol. The products were monitored at m/z 549 and 552, corresponding to molecular ions of the bis-TMS derivatives. 25-Hydroxylation was assayed after incubation of a 1:1 mixture of [6,7,7-²H₃]Chol and [25,26,26,26,27,27,27-²H₇]Chol. The 25-OH-Chol products were monitored at m/z 534 and 537, corresponding to the M-57 ion of the 3-TMS, 25-TBDMS derivative of 25-OH-Chol. 24-Hydroxylation was assayed after incubation of a 1:1 mixture of [23,23,24,24,25-²H₅]Chol and [25,26,26,26,27,27,27-²H₇]Chol. These analyses ignore possible effects of secondary isotope effects and, in the case of the 26-hydroxylation, of primary isotope effects in the further metabolism of 26-OH-Chol to the 26-acid.

Girotti et al. (339) studied lipid peroxidation in resealed human erythrocyte ghosts. Treatment of the erythrocyte ghosts with xanthine oxidase and xanthine was reported to give two hydroperoxides of Chol which, after treatment of the lipid extract with sodium borohydride, gave two polar sterols, ascribed to the 7-hydroxy and 7-hydroxy derivatives of Chol on the basis of TLC. An additional component of higher R_f was observed but not characterized. The addition of either catalase or superoxide dismutase was reported to prevent the formation of the Chol oxidation products when added to the xanthine oxidase plus xanthine-treated preparations. These materials were not observed in incubations lacking either xanthine oxidase or xanthine. Photo-oxidation of the erythrocyte ghosts in the presence of either rose bengal or protoporphyrin IX was reported to yield 5-hydroperoxy-5-cholest-6-en-3-ol, which was detected on the basis of the TLC behavior of the product of the borohydride reduction of the lipid extract of the oxidized membranes. In a subsequent study by the same laboratory, Bachowski et al. (44) studied the oxidation products of [4-¹⁴C]Chol incorporated into erythrocyte membrane preparations. Photo-oxidation of the labeled Chol was carried out in the presence of a hematoporphyrin derivative. Under these conditions, the major product was reported to be 5-hydroperoxy-5-cholest-6-en-3-ol for which characterization was limited to TLC and the mobility of the corresponding 5-hydroxysterol obtained upon borohydride reduction. After photo-oxidation, further incubation with ascorbate (1 mM) and FeCl₃ (10 µM) for 1 h in the dark led to a much more complex mixture of Chol oxidation products [including material ascribed to the 7- and 7-hydroperoxides of Chol (plus possibly 7-keto-Chol) and the 7- and 7-hydroxy derivatives of Chol], a complexity that was prevented by the addition of EDTA (50 µM).

Sevanian and McLeod (952) studied the oxidation of [4-¹⁴C]Chol in unilamellar liposomes containing Chol and phospholipids as induced by incubation under common autoxidation conditions, by oxidation with cumene hydroperoxide, or by -ray irradiation. The formation of the 5,6- and 5,6-epoxides of Chol, 7-keto-Chol, the 7-hydroperoxides of Chol, and the 7-hydroxy derivatives of Chol, 5,6-diOH-Chol, and cholesta-3,5-diene-7-one was reported. It should be noted that control preparations of the liposomes contained significant amounts of Chol oxidation products, which presumably arose during the preparations of the liposomes. The formation of oxidation products of Chol was lowest in liposomes prepared from a phospholipid containing saturated fatty acids (i.e., dipalmitoylphosphatidylcholine). Lang and Vigo-Pelfrey (542) studied the oxidation of Chol in small unilamellar vesicles and multilamellar vesicles composed of Chol and phosphatidylcholines (of variable degrees of unsaturation, i.e., native, egg PC, partially hydrogenated egg PC, or fully hydrogenated egg PC) after incubation at 50°C for 3 mo. As might be anticipated, oxidation of Chol was markedly suppressed or absent in the vesicles prepared from fully hydrogenated egg PC. The major oxidation product of Chol detected by HPLC was 7-keto-Chol, which was accompanied by lesser amounts of 7-OH-Chol and 7-OH-Chol. Significant amounts of unidentified products were noted in some experiments. Van Amerongen et al. (1150) studied the transfer of oxysterols from a mixed monolayer to acceptor vesicles in the subphase. With the use of labeled sterols, the transport of Chol was negligible, whereas notable transport (in order of increasing rate of transport) of 7-keto-Chol, 7-OH-Chol, and 25-OH-Chol was observed. Addition of the nonspecific lipid transfer protein (sterol carrier protein 2) increased the rate of transfer of Chol and of each of the oxysterols.

	IV. OCCURRENCE AND LEVELS OF OXYSTEROLS

Top Previous Next References

The occurrence and levels of oxysterols in plasma are topics of considerable potential importance in considerations of their potential physiological importance. Studies of this matter are complicated by difficulties in the separation and identification of the various oxysterols, especially in the absence of a comprehensive set of authentic standards. Quantitation of the sterols requires attention to recoveries in extractions, completeness of derivatization reactions, and the stability of the compounds to reactions used in the isolation and analytical procedures. Because a substantial fraction of a number of oxysterols is esterified to fatty acids, conditions of saponification are critical, especially in the case of 7-keto-Chol, which has been shown to undergo substantial decomposition under usual saponification conditions (273, 521, 603, 771). The ease of autoxidation of Chol, present at relatively high levels in plasma of normal human subjects, provides an additional problem, i.e., artifactual generation of oxysterols from Chol during sample storage, processing, and analysis. Certain oxysterols of biomedical interest (e.g., 26-OH-Chol, 24-OH-Chol, and 22-OH-Chol) are generally considered to not represent significant products of the autoxidation of Chol. Others (including 7-keto-Chol, 7-OH-Chol, 7-OH-Chol, 25-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, and 5,6-diOH-Chol) are recognized products of Chol autoxidation. More or less rigorous studies (see below) have indicated that some oxysterols of biomedical interest occur in fresh normal human plasma at levels of ~0.010-0.100 µM. To obtain valid estimates of the levels of such compounds, very major suppression or total elimination of autoxidation of Chol (present in plasma samples of normocholesterolemic subjects at ~5,000 µM) is essential. Formation of an oxysterol by autoxidation of Chol to the extent of only 0.001% could severely compromise a valid assay of a given oxysterol. Autoxidation of Chol can be reduced by analysis of fresh samples clearly free of hemolysis, inclusion of antioxidants, use of peroxide-free degassed solvents (and avoidance of solvents prone to peroxide formation such as ethyl ether), conduct of saponification and extraction steps under conditions involving rigorous exclusion of oxygen, and isolation (free of Chol) at an early stage in the analysis of the oxysterols. Even with such efforts to suppress autoxidation of Chol, an internal standard should be included in the assay of each sample to detect and quantitate the artifactual formation of each oxysterol from Chol during sample processing and analysis. To our knowledge, only one study (521) included such controls in the analysis of oxysterols in plasma.

In 1989, Kudo et al. (521) developed methodology based on the ability to separate the acetate derivatives of an number of oxysterols by the combination of reverse- and normal-phase HPLC. Fresh plasma samples were saponified and extracted with methyl t-butyl ether (an ether relatively resistant to peroxide formation) in a closed all-glass system under argon. The NSL were subjected to reverse-phase MPLC to isolate the polar sterols free of Chol. Acetylation of the polar sterols with [³H]acetic anhydride permitted not only following the separation of the oxysterols but also, with knowledge of the specific activity of the acetic anhydride, quantitation of the individual oxysterols. A critical feature of the study was the use of an internal standard of highly purified (immediately before use) [¹⁴C]Chol of high specific activity that was added to the plasma sample before any processing. Thus, by determination of the amounts of ³H and ¹⁴C associated with the acetate derivative of a given oxysterol, the amount of the oxysterol could be calculated as well as the amount formed artifactually from Chol during sample processing. The results of these studies demonstrated (after correction for artifactual generation of oxysterols by autoxidation during sample processing or analysis) the presence of significant levels of 26-OH-Chol, (24S)-24-OH-Chol, and 7-OH-Chol (Table 1). After correction for their formation by autoxidation of Chol, essentially none of the following sterols was observed in plasma: 7-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 5,6-diOH-Chol, and the 25-, 22R-, 20-, and 19-hydroxy derivatives of Chol. The levels of 26-OH-Chol (0.158 and 0.246 µM) and of (24S)-24-OH-Chol (0.074 and 0.107 µM) in the plasma of the two normal human subjects studied were considerable. The stereochemistry at C-25 of the 26-OH-Chol was not established. It is important to note that (24RS)-24-OH-Chol (469), (25R)-26-OH-Chol (290, 1103), and (25S)-26-OH-Chol (1103) have been shown to be highly active in lowering the levels of HMG-CoA reductase activity in cultured mammalian cells. For example, the (24RS)-24-OH-Chol caused a 50% suppression of HMG-CoA reductase activity in mouse L cells at 0.3 µM (469), and the 25R- and 25S-isomers of the 26-OH-Chol caused a comparable lowering of reductase activity at 0.26 and 0.16 µM, respectively, in the L cells (1103). The results of Kudo et al. (521) and studies by others (273, 446, 447, 505, 1011) indicate that the levels of 26-OH-Chol in plasma (or serum) are comparable to those shown to cause a substantial reduction in the levels of HMG-CoA reductase activity in cultured cells.

Other studies, none of which employed controls for autoxidation during processing of the samples, have reported the presence of the following oxysterols in human plasma: 5,6-epoxy-Chol (one or both isomers) (86, 122, 273, 348, 956), 7-OH-Chol (51, 97, 121, 122, 273, 505, 722, 1011, 1156), 7-OH-Chol (97, 122, 273, 505, 956, 1068, 1156), 7-keto-Chol (81, 122, 273, 446, 883, 956), 24-OH-Chol (121, 128, 273, 1011), 25-OH-Chol (121, 122, 273, 1011), 26-OH-Chol (121, 122, 128, 273, 375, 446, 447, 505, 1011, 1156), 4-OH-Chol (121, 128), and 5,6-diOH-Chol (122, 273, 956). A review of earlier published studies on this matter is included in the article of Kudo et al. (521) on oxysterols in human plasma. In one early study, Smith et al. (1011) reported the presence of six oxygenated oxysterols in the sterol esters of pooled human plasma. The concentrations of the individual oxidized sterols of relatively fresh plasma were estimated to be from 5 to 106 ng/ml. Analysis of stored samples of plasma indicated that the concentrations of 4 of the 6 sterols were markedly higher, 83 and 91 times higher for the cases of 7-keto-Chol and 7-OH-Chol. Smith et al. (1011) reported the presence of 25-OH-Chol in the ester fraction of fresh (5 ng/ml) and stored (20 ng/ml) samples of human plasma. In the study of Kudo et al. (521), which employed controls on autoxidation during sample processing and analysis, 25-OH-Chol was not detected in the plasma of two human subjects (with limits of detection of less than 3.2 and 0.4 ng/ml in the two cases). Other workers (121, 273, 375, 447, 505) also noted the absence or extremely low levels of 25-OH-Chol in human serum or plasma. Kou and Holmes (509) were unable to detect 25-OH-Chol in normal rat plasma; however, the estimated detection limit (~10 ng/ml) was quite high.

Harik-Khan and Holmes (375) reported on the levels of 26-OH-Chol in plasma of normal subjects [7] and those [18] with "angiographically proven atherosclerosis." The normal subjects were reported to show a mean value of 0.44 ± 0.15 (SD) µM with a range of 0.31-0.73 µM. The levels of 26-OH-Chol in 16 of the patients were reported to be within the range of "normal values" with a range of 0.18-0.65 µM. The levels of 26-OH-Chol (0.79 and 0.87 µM) in two of the subjects were considered to be elevated. The authors interpreted their results as "indicating that high 26-OHC levels cannot be a major factor in the development of atherosclerosis" (since most of the patients had "normal" levels of 26-OH-Chol). The authors reported a positive correlation between the serum levels of 26-OH-Chol and Chol. In these studies, an internal standard of biosynthetic 26-OH-[1,2-³H]Chol ("1,000 dpm"; 931 mCi/mmol) was added before saponification. The NSL were passed through a C₁₈ Sep-Pak cartridge and then subjected to reverse-phase HPLC and then normal-phase HPLC (with detection at 210 nm). The 26-OH-Chol peak was collected, and its radioactivity was estimated. It should be noted that no other standards were used and that no precautions to avoid autoxidation were employed. It was stated that "the identity and purity of the peak in three different samples was confirmed by electron impact mass spectrometry and a comparison of the spectra with authentic 26-hydroxycholesterol. The absence of any molecular ions greater than m/z 402 and the similarity of the fragmentation patterns of the standard and the samples confirmed the purity of the peak." GC-MS was done using a short (7 m) DB-1 column. The major weakness of this work is the lack of presentation of results demonstrating the ability of the chromatographic system employed to resolve 26-OH-Chol from other oxygenated sterols. The labeled internal standard was prepared by incubating [1,2-³H]Chol (of unstated purity) with rat liver mitochondria followed by purification of the product by HPLC. No detailed evaluation of the radiopurity was presented (or the ability of the HPLC system used to provide separations of 26-OH-Chol from other oxygenated sterols).

Breuer and Björkhem (122) described methodology for the simultaneous analysis of a number of oxysterols (7-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 7-keto-Chol, 5,6-diOH-Chol, 25-OH-Chol, and 26-OH-Chol) in serum as their TMS derivatives by GC-MS using deuterium-labeled internal standards for each of the oxysterols. Their approach involved addition of BHT to blood before processing, addition of deuterium-labeled internal standards to serum, saponification (55°C for 45 min), and extraction of NSL with CHCl₃-methanol (2:1), reverse-phase column chromatography (which partially resolved the oxysterols from Chol), and finally GC-MS of the TMS derivatives of the oxysterol fraction with selective ion monitoring. Mean values for 19 normal subjects are presented in Table 1. It should be noted that the levels of the oxysterols reported in Reference 122 are erroneously presented as milligram per milliliter (rather than ng/ml) as can be noted by discussion of the results by the authors. Whereas the mean value for 26-OH-Chol was similar to that of Kudo et al. (521), the mean values for 7-OH-Chol, 7-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 25-OH-Chol, and 5,6-diOH-Chol were considerably higher. The higher values for the latter oxysterols most probably are a consequence of their artifactual generation from Chol during sample processing. Dzeletovic et al. (273) subsequently reported improvements and extension to cover 24-OH-Chol and 7-OH-Chol in the GC MS analysis of oxysterols in plasma over that described by Breuer and Björkhem (122). In this modification, BHT and the deuterated internal standards for each of the oxysterols were added to plasma. After saponification under mild conditions (22°C for 2 h), the NSL were subjected to solid-phase extraction to remove the bulk of the Chol and then analyzed by GC-MS as TMS ether derivatives. The modified procedure was applied to 31 normocholesterolemic subjects. The mean values for the various oxysterols are presented in Table 1. The mean level of 26-OH-Chol was higher in males (0.445 ± 0.100 µM) than in females (0.326 ± 0.077 µM). No effect of sex on the levels of other oxysterols or differences in the levels of Chol in males and females was noted. Some aspects of the methodology used in this study warrant further comment. The saponification was carried out at 22°C since it was found that 7-keto-Chol decomposed (under the same conditions) at 38 or 55°C. Furthermore, "when the hydrolysis time was increased from 1 to 14 h an almost 50% loss in 7-oxocholesterol was observed." Furthermore, it was stated that "the hydrolysis procedure was always performed under an argon atmosphere since higher oxysterol concentrations were obtained when the hydrolysis was performed in air." The quantitation of the various oxysterols was based on GC-MS analyses (involving selected ion monitoring) of the TMS ether derivatives and the use of deuterium-labeled internal standards that were added to the plasma before sample processing. No data were provided on the identity and purity of the various synthetic deuterium-labeled sterols with the exception of the statement that "the internal standards were pure as determined by GC-MS after the workup described unless otherwise stated." The latter qualification applied to a sample of [26,26,26,27,27,27-²H₆]-5,6-epoxy-Chol which was reported to contain ~5% [26,26,26,27,27,27-²H₆]-5,6-epoxy-Chol "as determined by GC-MS." No data were provided as to the determination of the location of the label in the sterols. These matters appear to be particularly important with regard to two of the deuterium-labeled oxysterols. The preparation of [23,23,24,25,25-²H₅]cholest-5-ene-3,24-diol was described (273). It should be noted that there can only be one hydrogen at C-25 in this sterol. No characterization of the product was reported. No data on the localization of the deuterium or the extent of deuteration were presented. The procedure described could be expected to yield [22,23,24,25-²H₄]-24-OH-Chol. The selected ion monitoring of the TMS derivative of the unlabeled and labeled 24-OH-Chol was carried out at m/z 413 and 416, respectively. The ion at m/z 413 corresponds to an anticipated loss of C₃H₇ (corresponding to loss of C-25, C-26, and C-27) plus the loss of TMSOH. However, the origin of the loss of the TMSOH was not established (from C-3 or C-24). In the case of the deuterium-labeled 24-OH-Chol, the number and location of the deuterium atoms become important in interpretation of the GC-MS data. The location of deuterium label at or near the site of the concerned major EI-induced fragmentations is undesirable. Moreover, evidence for the precise origin of the ion at m/z 416 in the deuterated 24-OH-Chol was not presented. Potential problems also exist with regard to the nature of the d₅-26-OH-Chol internal standard. It was stated that "this compound was prepared by Clemmensen reduction of kryptogenin in deuterated medium as described." The references cited were to the basic method, as reported by Scheer et al. (905), for the synthesis of 26-OH-Chol by Clemmensen reduction of kryptogenin. The second reference was to Wachtel et al. (1175), which deals with the preparation of ³H-labeled 26-OH-Chol by Clemmensen reduction of kryptogenin in the presence of tritiated water. The paper by Dzeletovic et al. (273) does not deal with the results of an important reinvestigation of the Clemmensen reduction of kryptogenin (479) and later of the Clemmensen reduction of diosgenin (710), which demonstrated the complexity of this procedure and of previously undescribed byproducts. For example, Kluge et al. (498) reported that both Clemmensen reduction of kryptogenin and Wolff-Kishner reduction of the resulting 22-ketosterol by-product led to epimerization at C-20 and C-25. As noted elsewhere (711), use of this approach requires careful attention to the purity of intermediates and products and the localization of the deuterium in the concerned sterols. It should also be noted that no assignment of the location of the deuterium was made. The authors noted that "the maximum theoretical number of deuterium atoms that can be incorporated in this molecule is 10 (in positions 15, 16, 17, 20, 22, and 23). However, molecular species with 5 atoms were most abundant. Probably these are a mixture of molecular species with 5 deuterium atoms in different positions." Also, in the previous publication (122) on the d₅-26-OH-Chol, a partial MS (m/z 400 to m/z 500) of its TMS ether derivative was presented along with comparable data on the undeuterated compound. No analysis or comparison was made; however, it is very clear that the deuterated sample gave a number of significant, discrete ions in the range m/z 470 to m/z 500, a region in which no ions were presented for the undeuterated sterol. Similarly, the deuterated sterol also gave a number of significant, discrete ions in other regions in which analogous ions were not present in the undeuterated sterol. These data raise question as to the identity and purity of the d₅-26-OH-Chol.

Breuer (121) reported on the levels of several oxysterols in plasma of normal human subjects. Notable was the indication of the presence of significant levels of 4-OH-Chol in plasma. The methodology was reported to be the same as in Reference 273. The identification of the 4-OH-Chol was based on results of GC-MS studies of its TMS derivative and that of a synthetic standard. The mean level of the 3,4-dihyroxysterol in eight normal human subjects was reported to be 0.090 ± 0.010 µM. The mean levels of other oxysterols observed (i.e., 26-OH-Chol, 25-OH-Chol, 24-OH-Chol, 7-OH-Chol, and 7-OH-Chol) are presented in Table 1. Significant levels of 26-OH-Chol (0.410 µM), 24-OH-Chol (0.177 µM), and 7-OH-Chol (0.092 µM) were observed. Especially notable are the very low levels of 25-OH-Chol and 7-OH-Chol, similar to that observed by Kudo et al. (521). Brooks and Cole (128) had previously noted, in abstract form, the presence of 4-OH-Chol in human serum. It was reported that "in some sera, 4-OH-Chol was detected in significant amounts (~20-600 ng/ml)." This would correspond to 0.05-1.49 µM. Full details were not presented. The general procedure involved extraction, saponification, "chromatographic isolation of minor sterol fractions," derivative formation, and GC-MS.

Lütjohann et al. (596) studied the levels of 24-OH-Chol in plasma obtained from eight human subjects. Sampling was made from brachial artery and internal jugular vein. The mean level of the 24-hydroxysterol in the arterial samples (0.147 ± 0.019 µM) was reported to be lower (P < 0.02) than that of the venous samples (0.165 ± 0.029 µM). Inspection of the graphical data presented showed major differences in two subjects, no difference in three subjects, and relatively slight differences in three subjects. The variation in individual subjects was not studied. The authors interpreted their results as demonstrating "a net flux" of 24-OH-Chol "from the brain into the circulation." A similar arterial-venous difference was not observed in the case of 7-OH-Chol, 7-OH-Chol, 7-keto-Chol, 25-OH-Chol, 26-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, or 5,6-diOH-Chol (although specific data on these sterols were not presented). Björkhem et al. (95) extended this research to include four additional subjects, one of which showed no arterial-venous difference for 24-OH-Chol. However, three of the four new subjects showed a substantially higher level of 24-OH-Chol in the peripheral artery than in the internal jugular vein. For the 12 total subjects, the mean arteriovenous difference was 10.2 ± 2.8 ng/ml (P = 0.004). Sampling was also made from the hepatic vein and the brachial artery of 12 other normal subjects. The mean levels of 24-OH-Chol in the arterial samples was found to be slightly higher than that for the venous samples (mean arteriovenous difference of 7.4 ± 2.2 ng/ml; P < 0.006), suggesting a net uptake of 24-OH-Chol by liver.

Lütjohann et al. (596) observed that the 24-OH-Chol in plasma was largely found as esters, i.e., ~71% as fatty acid esters and 11% as sulfate esters. The sulfate ester(s) was not directly determined but was estimated by determination of the sterol after treatment under conditions for the solvolysis of sulfate esters. The 24-OH-Chol in plasma (and in liver) was reported to be only the 24S-isomer on the basis of capillary GC studies. No diurnal variation in the levels of 24-OH-Chol in plasma was observed in two human subjects. The ratios of 24-OH-Chol to Chol in plasma were higher in children (157 ± 74 ng/mg) than in adults (35 ± 9 ng/mg). In contrast, the ratios of 26-OH-Chol to Chol were lower in children than in adults. It was stated that "there was little or no correlation between age and the levels of other circulating oxysterols."

Babiker and Diczfalusy (41) reported on the levels of oxysterol in plasma from seven normal human subjects in the fasting state. The oxysterol levels were determined by GC-MS methodology as described previously from the same laboratory (273). Mean levels of nine oxysterols in plasma were reported (Table 1). 26-OH-Chol, 24-OH-Chol, and 7-OH-Chol represented the major oxysterols present in plasma. Much lower levels were observed for oxysterols known to arise from autoxidation of Chol (e.g., 7-keto-Chol and 7-OH-Chol).

Sevanian et al. (956) reported on plasma levels of certain oxysterols of normocholesterolemic human subjects (age, sex, and health status not specified). Methodology involved modified Folch extraction, solid-phase extraction, saponification, treatment with diazomethane, TMS ether formation, and GC-MS with selected ion monitoring. BHT was added before extraction of the plasma but not before its storage (up to 2 mo at 70°C). Whereas the range of plasma Chol levels was from 171 to 222 mg/dl, the mean value was reported as 109 mg/dl. The reported mean levels of oxysterols were very high (Table 1). The number of subjects studied was not given. The only expression of variation presented was the range of values, which, for most of the oxysterols, was quite small. The levels of 7-keto-Chol, 7-OH-Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, and 5,6-diOH-Chol were very much higher than those reported by others. In addition to the oxysterols presented in Table 1, a high mean value (3.4 µM) was reported for cholesta-3,5-dien-7-one. The authors stated that "analysis of cholesterol, freshly purified by HPLC and collected under argon and then subjected to the isolation procedure described for plasma samples, indicated that formation of cholesterol oxides from cholesterol during sample work-up was below the limits of quantitation." The representative chromatograms of standards presented were not impressive; the TMS derivative of 7-OH-Chol was incompletely separated from the TMS ether of Chol, and the TMS derivative of 7-OH-Chol was incompletely separated from the TMS ether of 5,6-epoxy-Chol. However, selective ion monitoring was used for these oxysterols. A representative chromatogram for human plasma was also not impressive. No peaks ascribable to 26-OH-Chol or 24-OH-Chol were reported.

Mol et al. (662) also studied the levels of oxysterols in plasma from normal human subjects. Mean values for 13 subjects are presented in Table 1. No values were reported for 7-OH-Chol, 5,6-epoxy-Chol, 24-OH-Chol, 26-OH-Chol, or 4-OH-Chol. Methodology cited included Folch extraction, saponification of the total lipids, silica gel column chromatography, and GC-MS analyses of the TMS derivatives of the sterols. No labeled internal standards were used in the GC-MS studies. BHT was added to the plasma before storage. Samples were stored "at 80°C for up to 12 months" before analysis. It should be noted that the mean level of 7-OH-Chol reported in this study is higher than those reported by others (41, 121, 273, 521). The reported mean level of 7-keto-Chol is considerably higher than that reported by Dzeletovic et al. (273) and Babiker and Diczfalusy (41). It is very likely that the levels of oxysterols reported by Mol et al. (662) are artifactually elevated due to autoxidation of Chol during sample storage and/or processing. Treatment of the subjects with vitamin E for 4 wk was reported to have no effect on the levels of the oxysterols studied. Non-insulin-dependent diabetic subjects (n = 10) showed no differences in the levels of the oxysterols studied relative to those in the control subjects. Higher mean levels of 5,6-epoxy-Chol and 7-keto-Chol were reported in cigarette smokers than in the control subjects.

One recent study (890) claimed that the levels of 7-OH-Chol were significantly associated with progression of carotid atherosclerosis. However, another study (1160) noted no association between the degree of coronary stenosis and the levels of unesterified 7-OH-Chol (or any other oxysterol studied) in plasma. These studies are considered in more detail in section IX.

Emanuel et al. (277) investigated the levels of "Chol oxidation products" (i.e., the combination of 5,6-epoxy-Chol, 5,6-epoxy-Chol, 7-OH-Chol, and 7-keto-Chol) in human plasma and in chylomicrons at various early times after the administration of a test meal of spray-dried powdered eggs (0.7 g/kg body wt) in the form of scrambled eggs. The Chol oxidation products and their levels (µg/g) were as follows: 5,6-epoxy-Chol, 50; 5,6-epoxy-Chol, 90; 7-OH-Chol, 60; and 7-keto-Chol, 30. The methodology was apparently as follows. Plasma was subjected to Folch extraction. The extracts were apparently saponified to give NSL (conditions not given) that were silylated and then analyzed by capillary GC. An illustrative chromatogram obtained from plasma samples obtained from one individual showed very poor resolution. Assignment of peaks (in order of elution) were Chol, 5,6-epoxy-Chol, 5,6-epoxy-Chol, 7-OH-Chol, and 7-keto-Chol. However, a number of other peaks were present for which no comments were made. The separations were not such as to provide confidence for quantitation. The authors claimed increases in the "total Chol oxidation products" in plasma after ingestion of the test meal. Little or no increases were observed in plasma after the ingestion of a comparable test meal prepared from fresh eggs. In most (but not all) of the subjects, apparent peaks of "total Chol oxidation products" in plasma and in chylomicrons isolated from plasma occurred at between 2.75 and 4 h after administration of the test meal prepared from the powdered egg material. It is important to note that data beyond 4 h were not presented. In some individuals, the levels of total "Chol oxidation products" rose as much as ~15 mg/l. With the assumption of a molecular weight of 402, this would correspond to ~37 µM.

Very high levels of 26-OH-Chol in plasma from baboons have been reported in three studies from one laboratory (376, 526, 527). Hasan and Kushwaha (376) reported on the plasma levels of 26-OH-Chol in baboons on a chow diet; the animals had previously been established to be "high responders" (n = 6) or "low responders" (n = 6) to dietary Chol. The mean levels (±SE) of 26-OH-Chol in the two groups on the chow diet were reported to be 26.6 ± 9.2 and 27.9 ± 5.2 µM, respectively. The same authors also reported on the levels of 26-OH-Chol in plasma from other baboons classified as high responders (n = 9) and low responders (n = 9) to dietary Chol while maintained on a high-fat (40% of calories from lard), high-Chol (1.7 mg/kcal) diet. Even higher levels of 26-OH-Chol in plasma were reported for animals on the high-fat, high-Chol diet. The mean level in low responders (167 ± 62 µM) was reported to be higher than those of the high responders (42.5 ± 2.4 µM). From the data presented, the percentages of 26-OH-Chol in Chol plus 26-OH-Chol in the low responders and high responders were 4.3 and 0.6%, respectively. In a subsequent study using the same methodology, Kushwaha et al. (526) studied the levels of 26-OH-Chol in young male baboons on a chow diet (0.03 mg Chol/kcal) and upon administration of a high-fat (40% of calories from coconut oil), high-Chol (1.35 mg/kcal) diet. On the basis of the response to the high-Chol, high-fat diet with respect to total plasma Chol and the levels of LDL + VLDL Chol, the animals were divided into three groups: high responders (n = 5), intermediate responders (n = 4), and low responders (n = 5). Plasma levels of 26-OH-Chol were determined at 0, 1, 3, 6, 10, 18, 26, 36, 52, 78, and 104 wk. The results were presented in graphical form only with comments in the text. The levels of 26-OH-Chol in plasma of the low responders were significantly higher than those in the high responders at 1, 3, 6, and 10 wk. The levels of 26-OH-Chol on the high-fat, high-Chol diet were higher than the mean value on the chow diet at weeks 3, 6, and 10. On changing from the chow diet to the high-fat, high-Chol diet, the level of 26-OH-Chol in plasma of low responding baboons increased and reached a maximal level (~100 µM) at 3-10 wk and thereafter decreased to ~50 µM for the remainder of the period of study. In the high responders, changing from the chow diet to the high-fat, high-Chol diet resulted in little or no effect on the levels of 26-OH-Chol in plasma, with mean values of ~30-40 µM throughout the period of study. In another study (527) from the same laboratory, the mean levels of 26-OH-Chol in the plasma of six ovariectomized baboons on a high-Chol (1.7 mg/kcal), high-fat (40% of calories from lard) diet were reported to be 9.9 ± 5.7 µM. Although the mean plasma level of 26-OH-Chol of progesterone-treated animals was not significantly different from controls, treatment with estrogen (estradiol cypionate) or the combination of estrogen and progesterone was associated with increased levels of 26-OH-Chol in plasma (21.0 ± 2.5 and 34.9 ± 6.2 µM, respectively). Plasma levels of 26-OH-Chol were reported to show a negative correlation with total plasma Chol and LDL Chol levels.

The reported levels of 26-OH-Chol in plasma from baboons in these studies are very much higher than those reported in credible studies for human subjects (Table 1). The methodology used in the baboon studies was stated to be based on the HPLC method of Harik-Khan and Holmes (375). Plasma samples were "saponified with a mixture of NaOH/methanol (1:9) for an hour at a constant pressure of 15 psi" (376). The temperature was not specified; however, in a subsequent publication (526), mention of heating under reflux was made. The saponification conditions of Harik-Khan and Holmes (375) involved methanol-3 M NaOH (9:1) at 37°C overnight with shaking. In the latter study (375), an internal standard of 26-OH-[³H]Chol was employed. In contrast, the baboon studies employed 7-keto-Chol as an internal standard that was added to the saponified mixture along with hexane and water. The mixture was centrifuged, and the hexane layer containing the 26-OH-Chol was "collected and dried under nitrogen." The resulting sample was dissolved in methanol and "injected to a C₁₈ reverse-phase silica column" (precise column and dimensions not given) using methanol-water (90:10) as the solvent; components were detected by absorbance at 210 nm. The identification of 26-OH-Chol was based solely on its retention time (7-8 min). However, only two other standards were mentioned: 7-keto-Chol (15 min) and Chol (60 min). The use of 7-keto-Chol as an internal standard presents notable problems. First, 7-keto-Chol could be present in the plasma sample or formed from Chol during sample storage and/or processing. Second, the addition of the 7-keto-Chol to the strongly alkaline solution could lead to its decomposition (273, 521, 603, 771). The estimation of the level of the 7-keto-Chol is of critical importance, since the quantitation of the 26-OH-Chol is based on an accurate and valid determination of the 7-keto-Chol. Also, as noted above, the mobilities of other oxygenated sterols potentially present in the sample were not established, and the chemical nature of the material with the retention time of 26-OH-Chol in this HPLC system was not established. It should also be noted that the only chromatography employed was simple reverse-phase HPLC that did not correspond to the procedures utilized by Harik-Khan and Holmes (375), i.e., solid-phase extraction, reverse-phase HPLC, and normal-phase HPLC.

In a subsequent study (190) from the same laboratory, substantially lower plasma levels of 26-OH-Chol were reported (without comment). The oxysterol levels were stated to have been measured by the same HPLC methodology as used previously (526) with the exception of a modification in which esterified oxysterols were hydrolyzed with Chol esterase. No details on this modification were provided. In this study, the high-Chol, high-fat diet contained 0.45 mg Chol/kcal and 40% of calories from coconut oil. The basal diet was low in Chol (0.03 mg/kcal) and fat (10% of total calories). The levels of 26-OH-Chol in the plasma of low responding (n = 6) and high responding (n = 6) baboons on the basal, low-Chol diet were reported as 0.130 ± 0.030 µM for the low responders and 0.126 ± 0.013 µM for the high responders. Upon changing to the high-Chol, high-fat diet, the levels of 26-OH-Chol in plasma of the low responders at 3, 6, 10, and 18 wk were reported as 0.237 ± 0.019, 0.251 ± 0.036, 0.180 ± 0.014, and 0.202 ± 0.018 µM, respectively. The levels of 26-OH-Chol in plasma from the high responders at the same time points were reported as 0.162 ± 0.011, 0.142 ± 0.008, 0.148 ± 0.019, and 0.177 ± 0.009 µM. On the high-Chol, high-fat diet, the levels of plasma 26-OH-Chol in the high responders did not differ significantly from the mean values on the basal (low-Chol, low-fat diet). In the low responders, the mean levels of 26-OH-Chol at 3, 6, and 18 wk were significantly higher than that on the basal diet. On the high-Chol, high-fat diet, the mean levels of 26-OH-Chol in the low responders were significantly higher than the high responders at 3 and 6 wk, but not significantly different at 10 and 18 wk. On the basal diet, no correlation was reported between plasma 26-OH-Chol levels and plasma lipoprotein Chol levels. On the high-Chol, high-fat diet, a significant negative correlation was observed at 3 and 6 wk between the plasma levels of 26-OH-Chol and LDL + VLDL Chol levels.

The results of recent studies of the levels of 26-OH-Chol in baboons fed either a chow diet or a high-fat, high-Chol (1.7 mg/kcal) diet by reverse-phase HPLC of the NSL followed by GC-MS (using an internal standard of deuterium-labeled 26-OH-Chol) gave results quite different from those described above (unpublished data). On the chow diet, mean plasma 26-OH levels (±SD) were 0.188 ± 0.025 µM (n = 7) in females and 0.142 ± 0.047 µM (n = 16) in males. On the high-fat, high-Chol diet, mean plasma levels for plasma 26-OH-Chol were 0.262 ± 0.070 µM (n = 6) for females and 0.294 ± 0.033 µM (n = 4) for males. From the results of all of the animals on each of the diets, a positive correlation was observed between the levels of 26-OH-Chol and Chol in plasma.