Inhibitors of sterol synthesis. Chromatography of acetate derivatives of oxygenated sterols

The separation of the acetate derivatives of a number of oxygenated sterols was achieved by medium pressure liquid chromatography on silica gel columns and by normal and reversed phase high performance liquid chromatography. We have explored the application of these chromatographic systems for the analysis of oxygenated sterols of plasma samples from two normal human subjects. The addition of highly purified [14C]cholesterol to plasma permitted the detection and quantitation of oxygenated sterols formed by autoxidation of cholesterol during processing of the samples. Special attempts to suppress autoxidation of cholesterol included the use of an all-glass closed system for saponification and extraction under argon followed by rapid removal of cholesterol from the polar sterols by reversed phase medium pressure liquid chromatography. Chromatographic analyses of the [3H]acetate derivatives of the polar sterols provided a sensitive approach for the detection and quantitation of the individual oxygenated sterols. Oxygenated sterols detected in plasma included cholest-5-ene-3 beta,26-diol, (24S)-cholest-5-ene-3 beta,24-diol, and cholest-5-ene-3 beta,7 alpha-diol. After correction for their formation by autoxidation of cholesterol during processing of the samples, very little or none of the following sterols were observed: cholest-5-ene-3 beta,7 beta-diol, 5 alpha,6 alpha-epoxy-cholestan-3 beta-ol, 5 beta,6 beta-epoxy-cholestan-3 beta-ol, and cholestane- 3 beta, 5 alpha,6 beta-triol, and the 25-hydroxy, 22R-hydroxy, 21-hydroxy, 20 alpha-hydroxy, and 19-hydroxy derivatives of cholesterol.     

Pro-Oxidant Action of Superoxide Dismutase in the Autoxidation of Rifamycin Sv

6-ketocholestanol. a naturally occurring oxygenated sterol, when incubated with human neutrophils (PMN), can inhibit superoxide and hydrogen peroxide generation in a dose-dependent fashion. This is accompanied by inhibition of stimulated PMN aggregation without alteration in cellular viability. This inhibitory effect is not affected by washing of the cells, and cannot be blocked by the addition of free cholesterol to the medium. These data are consistent with prior observations which showed an inhibitory effect on PMN chemotaxis by certain oxygenated sterol compounds. and support the hypothesis that certain oxygenated sterols can affect a variety of human PMN functions by a mechanism that may involve perturbation of the plasma membrane.     

Metabolic effects of plant sterols and stanols (Review)

High serum LDL cholesterol concentration is a major risk factor for cardiovascular complications. This risk can be lowered by diet. In this respect foods containing plant sterol or stanol esters can be useful for mildly- and hypercholesteraemic subjects. Plant sterols and stanols, which are structurally related to cholesterol, decrease the incorporation of dietary and biliary cholesterol into micelles. This lowers cholesterol absorption. Furthermore, these components increase ABC-transporter expression, which may also contribute to the decreased cholesterol absorption. Consequently, cholesterol synthesis and LDL receptor activity increase, which ultimately leads to decreased serum LDL cholesterol concentrations. Animal studies have further shown that these dietary components may also lower atherosclerotic lesion development. Plant sterols and stanols also lower plasma lipid-standardized concentrations of the hydrocarbon carotenoids, but not those of the oxygenated cartenoids and tocopherols. Also, vitamin A and D concentrations are not affected. Although absorption of plant sterols and stanols (0.02-3.5%) is low compared to cholesterol (35-70%), small amounts are found in the circulation and may influence other physiological functions. However, there is no consistent evidence that plant sterols or stanols can change the risk of colon or prostate cancer, or immune status. In conclusion, plant sterols and stanols effectively reduce serum LDL cholesterol and atherosclerotic risk. In addition potential effects of plant sterols and stanols on other metabolic processes remain to be elucidated.     

Biosynthesis of squalene and sterols by rat aorta

The synthesis of nonsaponifiable compounds from radioactive mevalonate by segments of adult rat aorta was studied in vitro. The labeled products consisted largely of substances with the chromatographic and chemical behavior of squalene, lanosterol, lathosterol, and cholesterol. Even after 3 or 4 hr of incubation, the incorporation of mevalonate into squalene was higher than its incorporation into C(27) sterols; cholesterol contained less than 20% of the radioactivity in the total sterols. Lanosterol was the most highly labeled sterol. The level of radioactivity in lathosterol was comparable to the level in cholesterol. Small amounts of radioactivity were found in other sterols. Material with the same mobility on TLC as 7-dehydrocholesterol had less radioactivity than cholesterol, but more than sterols with the mobility of desmosterol. The results of measurements made after short periods of incubation showed that squalene and lanosterol became labeled before the other nonsaponifiable compounds.     

植物甾醇·甾烷醇及其降血清胆固醇的作用

植物甾醇.甾烷醇在自然界广泛分布,以游离型、脂肪酸酯或糖苷形式存在。植物甾醇.甾烷醇能抑制小肠内胆固醇的吸收,具有降低血清胆固醇的作用。由于其来源于天然植物,且吸收率很低,因此具有很高的安全性。本文对植物甾醇.甾烷醇的降血清胆固醇作用做一综述。     

Distribution and dynamic state of sterols and steroids in the tissues of an insect, the roach Eurycotis floridana

The total concentrations of sterols in the tissues of the roach, Eurycotis floridana, reared under aseptic conditions and on semisynthetic diets, are similar to, but somewhat lower than, those of tissues of vertebrates. Total concentrations of tissue sterols are relatively independent of dietary concentration of sterols whether the diet contains 0.1% cholesterol as the sole sterol, or a "minimal cholesterol" mixture (0.1% cholestanol together with 0.005% cholesterol). Under the latter conditions the cholesterol is incorporated preferentially into most tissues and remains almost exclusively unesterified, while the cholesterol-sparing sterol is esterified to varying degree, depending upon the tissue. The turnover of tissue sterols has been studied. Cholesterol of the tissues of adult insects grown on a diet containing this sterol alone may be displaced by cholestanol fed as 5% of the total diet, initially at an appreciable rate but later much less rapidly. In growing insects that have received a diet containing cholestanol together with minimal cholesterol, the unesterified cholesterol turns over slowly in all tissues and immeasurably slowly in some. The unesterified sparing sterol, on the other hand, turns over at a much greater rate. The turnover of sterols during growth is accompanied by a shift of sterols from the unesterified to the esterified pool in all tissues. The fat body of the growing insect stores sterols (apparently as their esters) that have been displaced from other tissues. The fat body of the adult does not show evidence of sterol storage. Polar derivatives of sterols are present in minor amount in all tissues of the insect, most abundantly in the mid-intestine and gastric caeca. These compounds seem likely to be C(27) steroids.     

Effect of oxygenated sterols on 3-hydroxy-3-methylglutaryl coenzyme a reductase and DNA synthesis in phytohemagglutinin-stimulated human lymphocytes

Fifteen oxygenated sterols at the concentration of 25 μg/ml were tested on DNA synthesis of phytohemagglutinin stimulated human lymphocytes. In a cholesterol containing medium, the inhibitory effect was strictly dependent of the side chain structure of the sterol and only due to an hydroxylation at position 25. Three oxygenated sterols, which slightly inhibited DNA synthesis, strongly suppressed the peak of 3-hydroxy-3-methylglutaryl CoA reductase activity that normally precedes DNA synthesis. The 25-hydroxycholesterol suppressed the reductase activity even at 5 μg/ml, but was active on DNA synthesis only at 25 μg/ml; at this concentration, the later the 25-hydroxycholesterol was added, the weaker the inhibition of DNA synthesis was. Hence the sterol synthesis related to the early increase of 3-hydroxy-3-methylglutaryl CoA reductase activity is probably not essential to the cellular division. Several hypothesis on the mechanism of action of the 25-hydroxycholesterol are discussed.     

DNA synthesis and 3-hydroxy-3-methylglutaryl CoA reductase activity in PHA stimulated human lymphocytes: A comparative study of the inhibitory effects of some oxysterols with special reference to side chain hydroxylated derivatives

The effects of a series of oxygenated sterols on DNA synthesis and HMG-CoA reductase activity were tested in human lymphocytes. The cells were stimulated by PHA and cultured in cholesterol containing medium. The inhibitory effects of sterols on DNA synthesis were strictly related to the position and the configuration of the hydroxyl on the side chain, to the side chain conformation and integrity and to the structure of the sterol nucleus. The inhibition of HMG-CoA reductase activity was less dependent on these structural features since all the sterols tested were strong inhibitors. In our experimental conditions the inhibition of DNA synthesis was not related to the suppression of the HMG CoA reductase activity. The specificity of the structures required for DNA synthesis inhibition could be explained by the involvement of a specific hydroxysterol binding protein.     

Photoaffinity labeling of the oxysterol receptor

A cytosolic receptor protein for oxygenated sterols, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol biosynthesis, can be labeled covalently by photoactivation of 7,7'-azo-[5,6-3H]cholestane-3 beta,25-diol. Other compounds tested for their potential as photoaffinity reagents were: 25-hydroxycholesta-4,6-dien-3-one, 3 beta,25-dihydroxycholest-5-en-7-one, and 3 beta-hydroxycholesta-8(14),9(11)-dien-15-one. These sterols did not bind to the receptor with adequate affinity, were not readily photolyzed, or did not react covalently with the receptor during photolysis. The successful photoaffinity label, 7,7'-azocholestane-3 beta,25-diol, binds to the receptor with high affinity (Kd = 9.1 nM). After activation of the partially purified oxysterol-receptor complex with UV light (greater than 300 nm), several covalently labeled proteins were found upon sodium dodecyl sulfate-gel electrophoresis. Labeling of one protein, Mr approximately 98,000, was much reduced when the binding reaction was carried out in the presence of an excess of unlabeled oxysterol. Under the reaction conditions investigated so far, approximately 1% of the specifically bound sterol was covalently linked after photolysis. These results are consistent with previous information suggesting that the Mr of the receptor subunit is approximately 97,000. The covalent labeling of the receptor reported herein should facilitate its further purification and characterization.     

The use of the Dhcr7 knockout mouse to accurately determine the origin of fetal sterols

Mice with a targeted mutation of 3beta-hydroxysterol Delta(7)-reductase (Dhcr7) that cannot convert 7-dehydrocholesterol to cholesterol were used to identify the origin of fetal sterols. Because their heterozygous mothers synthesize cholesterol normally, virtually all sterols found in a Dhcr7 knockout fetus having a Delta(7) or a Delta(8) double bond must have been synthesized by the fetus itself but any cholesterol had to have come from the mother. Early in gestation, most fetal sterols were of maternal origin, but at approximately E13-14, in situ synthesis became increasingly important, and by birth, 55-60% of liver and lung sterols had been made by the fetus. In contrast, at E10-11, upon formation of the blood-brain barrier, the brain rapidly became the source of almost all of its own sterols (90% at birth). New, rapid, de novo sterol synthesis in brain was confirmed by the observation that concentrations of C24,25-unsaturated sterols were low in the brains of all very young fetuses but increased rapidly beginning at approximately E11-12. Reduced activity of sterol C24,25-reductase (Dhcr24) in brain, suggested by the abundance of C24,25-unsaturated compounds, seems to be the result of suppressed Dhcr24 expression. The early fetal brain also appears to conserve cholesterol by keeping cholesterol 24-hydroxylase expression low until approximately E18.     

Pinocytosis in L cells: its dependence on membrane sterol and the cytoskeleton

Pinocytosis in L-cells, grown in serum-free medium, was depressed when cultures were treated with oxygenated derivatives of cholesterol which inhibited sterol synthesis and reduced the sterol concentration of the plasma membranes. Noninhibitory sterols, such as cholesterol or desmosterol counteracted the effects of the inhibitors. Treatment with polylysine increased the rate of pinocytosis in sterol-depleted cells to a level similar to the enhanced rate found in polylysine treated control cells. Drugs which interfere with cytoskeletal systems (microfilaments, microtubules) also depressed pinocytosis but their effect could not be overcome by treatment with polylysine.     

Somatic cell genetics and the study of cholesterol metabolism

The regulation of cholesterol biosynthesis by extracellular cholesterol occurs both in whole animal tissue and in permanent somatic cell lines in culture. Permanent mammalian cells lines, under optimized growth conditions, are easily manipulated both biochemically and genetically. The Chinese hamster ovary cell line (CHO-K1) is the most widely used cell line for genetic studies. CHO-K1 is a pseudo-diploid mammalian cell exhibiting a short doubling time and a relatively high plating efficiency. Somatic cell mutants can be generated through mutagenesis and also by drug adaptation. Following mutagenesis, auxotrophs may be isolated either by selection or by screening. Most selection procedures for mutants of cholesterol metabolism must be done in serum depleted of cholesterol which requires the endogenous biosynthetic pathway to be intact. Mutants failing to produce cholesterol do not replicate their DNA and exhibit reduced concentrations of cholesterol in their membranes. BUdR and polyene antibiotics have both been used to select against the wild-type cells which incorporate these compounds and are killed, allowing the survival of the mutant cells. Both mevalonate and cholesterol auxotrophs have been isolated with the BUdR technique and have proven useful for elucidation of the early steps in cholesterol biosynthesis, particularly for the ratelimiting enzyme HMG-CoA reductase. Somatic cell fusion of a mutant and wild-type cell followed by chromosomal segregation, routinely used to map human genes, has also been used to map the human gene for HMG-CoA synthase. Such hybrids also provide valuable information on the dominance or recessivity of a specific lesion. DNA-mediated gene transfer into somatic cell mutants allows the selection of DNA sequences which complement the mutation, and is also useful for analysis of regions of regulatory significance. Mutants, resistant to the regulatory effects of oxygenated sterols, can be isolated following mutagenesis. Mutants of this type vary the lipid content of their membranes in response to cholesterol concentration in the medium. All such mutants tested exhibit a pleiotropic regulatory effect on more than one enzyme in the cholesterol biosynthetic pathway. Adaptation to drugs such as compactin and mevinolin, which inhibit HMG-CoA reductase, have been used to produce mutants which overexpress enzymes in the pathway. These amplified cells are useful sources of specific mRNAs for construction of cDNA libraries and gene isolation. Structure-function relationships of membrane sterols can be studied in cholesterol auxotrophs where changes in acyl-chain ordering can be manipulated by exogenous sterols in the medium.     

Comparison of the metabolism of cholesterol, cholestanol, and -sitosterol in L-cell mouse fibroblasts

The following data have been obtained from comparative studies on the metabolism of cholesterol, cholestanol, and beta-sitosterol by L-cell mouse fibroblasts. (1) When the sterols are added to the growth medium under similar conditions, cellular incorporation of cholesterol > cholestanol > beta-sitosterol; (2) only limited cellular esterification of these compounds occurs; (3) no metabolic products arising from the sterols could be detected; (4) influx of all sterols is dependent upon the concentration; and (5) exogenous cholesterol reduces mevalonate incorporation into cellular sterol to a lesser extent than acetate or glucose. The metabolism of these sterols is discussed in relation to their ability to influence de novo sterol biosynthesis.     

Evidence for the presence of 7-hydroperoxycholest-5-en-3 beta-ol in oxidized human LDL.

Low density lipoprotein (LDL) cholesterol is known to be oxidized both in vitro and in vivo giving rise to oxygenated sterols. Conflicting results, however, have been reported concerning both the nature and the relative concentrations of these compounds in oxidized human LDL. We examined the extracts obtained from Cu(2+)-oxidized LDL. Thin layer chromatography analysis showed that the sterol mixture became more complex with reaction time. Analysis of the components by thin layer chromatography and mass spectrometry allowed to establish that 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha OOH and beta OOH) are largely prevalent among the oxysterols at early times of oxidation. These hydroperoxy derivatives have not been previously identified in oxidized LDL. The concentration of 7-hydroperoxycholest-5-en-3 beta-ol decreased with oxidation time with a concomitant increase of cholest-5-en-3 beta, 7 alpha-diol (7 alpha OH), cholest-5-en-3 beta, 7 beta-diol (7 beta OH), cholesta-3,5-dien-7-one (CD) and cholest-5-en-3 beta-ol-7-one (7CO). After 24 h of oxidation a minor component of the LDL sterols was cholestan-3 beta-ol-5,6-oxide (EP).     

Analysis of oxysterol metabolomes

Oxysterols are oxygenated forms of cholesterol. This definition can, however, be expanded to include oxygenated derivatives of plant sterols and also of cholesterol precursors. Oxysterols are formed in the first steps of cholesterol metabolism and also from cholesterol by reactive oxygen species. Oxysterols were once thought of as simple intermediates, or side-products, in the conversion of cholesterol to hormonal steroids and bile acids, however, they have subsequently been shown to be biologically active molecules in their own right. In this article we will discuss methods of oxysterol analysis including "classical" gas chromatography-mass spectrometry (GC-MS) methods and more recent liquid chromatography (LC)-MS methods. Our main focus, however, will be on analytical methods based on "charge-tagging" and LC-tandem mass spectrometry (MS/MS or MS(n)) which we have developed over the last decade in our laboratory. Examples will be given of oxysterol analysis in brain, cerebrospinal fluid (CSF) and blood. The advantages and disadvantages of the various methods of oxysterol analysis will be discussed.     

Endogenous sterol synthesis is not required for regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by low density lipoprotein

It has been proposed that an endogenously synthesized oxysterol mediates the regulation of cholesterol biosynthesis by low density lipoprotein in cultured mammalian cells. Studies in this report demonstrate that under conditions in which squalene conversion to sterols is blocked either by inhibition of squalene cyclization or lanosterol demethylation, or both, low density lipoprotein regulates 3-hydroxy-3-methylglutaryl coenzyme A reductase normally. These observations rule out the hypotheses that either an endogenously synthesized oxygenated cholesterol biosynthetic intermediate or epoxysterol is required to mediate the inhibition of this enzyme by low density lipoprotein.     

Evidence for similarities and differences in the biosynthesis of fungal sterols

The sterol composition of two ascomycetous fungi, Saccharomyces cerevisiae and Gibberella fujikuroi, was examined by chromatographic (TLC, GLC, and HPLC) and spectral (MS and 1H-NMR) methods. Of notable importance was that both fungi produced cholesterol and a homologous series of long chain fatty alcohols (C22 to C30). In addition to ergosterol two novel sterols, ergosta-5,7, 9(11), 22-tetraenol and ergosterol endoperoxide, were isolated as minor compounds in growth-arrested cultures of yeast and in mycelia of G. fujikuroi. 24-Ethylidenelanosterol was also detected in mycelia of G. fujikuroi. A shift in sterol biosynthesis was observed by treatment with 24 (RS), 25-epiminolanosterol (an inhibitor of the S-adenosylmethionine C-24 transferase) and by monitoring the sterol composition at various stages of development. The results are interpreted to imply that the genes for 24-desalkyl, e.g., cholesterol, and 24-alkyl sterols, e.g., 24 beta- methyl cholesterol and 24-ethyl cholesterol, are distributed (but not always expressed) generally throughout the fungi but the occurrence of one or another compounds is influenced by the fitness (structure and amount) for specific sterols to act functionally during fungal ontogeny; sterol fitness is coordinated with Darwinian selection pressures.     

Occurrence of squalene and sterols in Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. Hester 1971.

The neutral lipid fraction of the photochromogenic, coryneform bacterium Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. contains the sterol precursor squalene and at least two sterols, cholesterol and beta-sitosterol. The compounds were characterized by mass spectrometry and combination gas-liquid chromatography--mass spectrometry. De novo sterol biosynthetic ability was shown from incorporation of 14C from D-[U-14C]glucose into squalene and the sterol fraction. The squalene concentration approximated 0.002 to 0.005% of the total dry cell weight, and the sterols approximated 0.03 to 0.05%.     

Cytotoxicity of cholesterol oxides and their effects on cholesterol metabolism in cultured human aortic smooth muscle cells

Incubation of monolayers of cultured human aortic smooth muscle cells with oxygenated sterols (25-hydroxycholesterol, 7-ketocholesterol, or cholesterol 5,6-epoxide) markedly inhibited growth though the viability of the culture was not affected. The effects on growth was concentration dependent, and 25-hydroxycholesterol was the most potent inhibitor of cellular growth as measured by decreased incorporation of thymidine into DNA and suppression of HMG-CoA reductase activity. The inhibitory effect of 25-hydroxycholesterol on cellular growth was not reversible if the cultures were grown in medium with normal fetal calf serum. However, in medium with delipidated serum, addition of purified cholesterol partially prevented growth inhibition induced by 25-hydroxycholesterol. Purified cholesterol, independently or in combination with tocopherol had no toxic effect on cellular growth. Addition of cholesterol oxides to the incubation medium stimulated lysosomal activation and release of acid phosphatase into the culture medium. The effect was concentration dependent and inversely related to cellular growth.