A novel olefinic rearrangement. The enzymic conversion of cholesta-7,9-dien-3β-ol into cholesta-8,14-dien-3β-ol

1. [3alpha-(3)H]Cholesta-7,9-dien-3beta-ol is converted in high yield into cholesterol by a 10000g(av.) supernatant fraction of rat liver homogenate. 2. Incubation of cholesta-7,9-dien-3beta-ol with [4-(3)H]NADPH and rat liver microsomal fractions under anaerobic conditions resulted in (3)H being incorporated into the 14alpha-position of cholest-7-en-3beta-ol. 3. Under anaerobic conditions in the absence of NADPH cholesta-7,9-dien-3beta-ol was isomerized into cholesta-8,14-dien-3beta-ol by rat liver microsomal fractions.     

Studies on delta8-delta7 isomerization and methyl transfer of sterols in ergosterol biosynthesis of yeast.

The formation of cholesta-7,24-dien-3 beta-ol and its activity as a substrate for the sterol side-chain methyltransferase in yeast have not previously been studied. Experiments with acetone-powder extracts of yeast showed that the sterol is formed from zymosterol by delta8-delta7 isomerization. However, direct conversion of cholesta-7,24-dien-3 beta-ol into zymosterol could not be demonstrated. The reversibility of the reaction was proved by the detection of 3H-incorporation into cholesta-8-en-3 beta-ol (with lathosterol as a carrier) from [3H]H2O in the medium. Incubation of cholesta-7,24-dien-3 beta-ol and S-adenosyl-L-[methyl-14C]methionine with the acetone-powder extract resulted in methylation of the sterol to form episterol. Similar incubation of zymosterol gave fecosterol and episterol, suggesting that fecosterol initially formed by the methylation was isomerized to episterol. In intact cells, however, an alternative pathway (zymosterol yields cholesta-7,24-dien-3 beta-ol yields episterol) may also operate. The relative importance of the two pathways is not known.     

Evaluation of the vitamin D activity of yeasts with altered sterol metabolism

The sterol content of Saccharomyces strains with altered ergosterol metabolism was studied by UV-spectrophotometry, thin-layer chromatography and chromatographic mass-spectroscopy. A technique for estimation of D-vitamin activity of the yeast strains is proposed. The irradiated biomass of the strains accumulated ergosta-5,7-dien-3 beta-ol and also cholesta-5,7,24-trien-3 beta-ol and cholesta-5,7,22,24-tetraen-3 beta-ol is characterized by high antirachitic activity.     

The induced biosynthesis of 7-dehydrocholesterols in yeast: potential sources of new provitamin D3 analogs.

The effect of low concentrations of a specifically designed sterol-24-transmethylase inhibitor, 25-aza-24, 25-dihydrozymosterol (10) on sterol production in Saccharomyces cerevisiae was examined. The synthesis of cholesta-5,7,22,24-tetraen-3beta-ol (4), its 7,22,24 analog (15) and the 7,24 analog (5) coupled with the availability of zymosterol (6) and cholesta-5,7,24-3beta-ol (3) derivatives facilitated a search for these sterols in cultures treated with this inhibitor. When S. cerevisiae was grown in the presence of 1.3 and 5 muM 10, it produced no ergosterol but accumulated zymosterol (6), cholesta-5,7,22,24-tetraen-3beta-ol (4) and related C27 sterols (3 and 5). These results indicate blockage of the side chain methylation that normally occurs during the biosynthesis of ergosterol in yeast by compound 10 is efficient. The cholesta-5,7,22,24-tetraen-3beta-ol is a close structural analog of provitamin D3 (7-dehydrocholesterol). The inhibited yeast thus provides a source of a potentially new provitamin D3 substitute.     

Mode of formation of cholesta-5,7-dien-3β-ol from cholest-5-en-3β-ol by larvae of Calliphora erythrocephala

1. The conversion of cholest-5-en-3beta-ol (cholesterol) into cholesta-5,7-dien-3beta-ol by axenic Calliphora erythrocephala larvae was demonstrated. 2. The transformation is probably direct (Delta(5)-->Delta(5,7)) and does not involve a Delta(0) intermediate (Delta(5)-->Delta(0)-->Delta(7)--> Delta(5,7)). 3. Delta(7)-bond formation involves the stereospecific elimination of the 7beta hydrogen atom. 4. The relative amounts of free and esterified sterols were determined in larvae grown on cholesterol as sole sterol source and on 5alpha-cholestan-3beta-ol supplemented with minimal amounts of cholesterol. 5. The significance of the results is assessed in relation to the probable role of cholesta-5,7-dien-3beta-ol as an intermediate in the biosynthesis of ecdysones.     

The formation and reduction of the 14,15-double bond in cholesterol biosynthesis

It was shown that 100mug quantities of 4,4'-dimethyl[2-(3)H(2)]cholesta-8,14-dien-3beta-ol (IIIa), tritiated cholesta-8,14-dien-3beta-ol, 4,4'-dimethyl[2-(3)H(2)]cholesta-7,14-dien-3beta-ol, dihydro[2-(3)H(2)]lanosterol and [24-(3)H]lanosterol were converted by a 10000g supernatant of rat liver homogenate into cholesterol in 17%, 54%, 6%, 9.5% and 24% yields respectively. From an incubation of dihydro[3alpha-(3)H]lanosterol with a rat liver homogenate in the presence of a trap up to 38% of the radioactivity was found to be associated with a fraction that was unambiguously shown to be 4,4'-dimethylcholesta-8,14-dien-3beta-ol. Another related compound, 4,4'-dimethylcholesta-7,14-dien-3beta-ol was also shown to be equally effective in its ability to trap compound (IIIa) from an incubation of dihydro[3alpha-(3)H]lanosterol. The mechanism of the further conversion of the compound (IIIa) into cholesterol occurred by the reduction of the 14,15-double bond and involved the addition of a hydrogen atom from the medium to C-15 and another from the 4-position of NADPH to C-14. Two possible mechanisms for the removal of the 14alpha-methyl group in sterol biosynthesis are discussed.     

The oxidation of Delta2, Delta2,4 and Delta4,6 steroids with RuO4

In order to find new ways for the functionalization of the A and B rings of the steroid nucleus, the reaction of 5alpha-androst-2-en-17beta-ol 17-acetate (1), cholesta-2,4-diene (4) and cholesta-4,6-dien-3beta-ol 3-acetate (7) was examined using stoichiometric amounts of ruthenium tetraoxide to yield 1,2-cis diols and/or alpha-hydroxy ketones. The reaction of 5alpha-cholest-2-en-3-ol 3-acetate (9) with ruthenium tetraoxide was also carried out and afforded, apart from an alpha-hydroxy ketone, also a diketone and a seco-dicarboxylic acid. The structures of all new steroids, including stereochemical details, were deduced by analysis of spectral data.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Solubilization and purification of steroid 8-isomerase

Steroid-8-ene isomerase that catalyzes isomerization of delta 8- to delta 7-sterols has been solubilized from rat liver microsomes with a mixture of two detergents, octylglucoside and sodium taurodeoxycholic acid. During a 40-fold enrichment of the solubilized enzyme, other enzymes of cholesterol biosynthesis, endogenous lipids, and electron carriers are removed. A comparison of properties of the solubilized and partially purified isomerase with the membrane-bound enzyme shows they are essentially identical with respect to pH profile, effect of inhibitors and cofactors, substrate specificity, and Km values. Addition of phospholipid to the partially purified enzyme stimulates activity as much as 1.8-fold over control rates. Although the relative rate of isomerization of cholesta-8,24-dien-3 beta-ol is six times that observed with cholest-8-en-3 beta-ol, the delta 8 to delta 7 ratio at equilibrium is approximately equal. The reversibility of the reaction has been demonstrated by the direct conversion of cholest-7-en-3 beta-ol to cholest-8-en-3 beta-ol; at equilibrium the delta 7-isomer is predominant (19/1). The purified enzyme does not catalyze isomerization of cholesta-8,14-dien-3 beta-ol and cholest-8(14)-en-3 beta-ol under conditions that result in equilibrium mixtures of isomers from cholest-8(9)-en-3 beta-ol. These results are consistent with the earlier suggestion that delta 8(14)-sterols are neither formed nor metabolized by the same microsomal enzymes that catalyze transformation of lanosterol to cholesterol.     

Sterol synthesis: studies of the metabolism of 14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol

[3 alpha-3H]14 alpha-Methyl-5 alpha-cholest-7-en-3 beta-ol has been prepared by chemical synthesis. The metabolism of this compound has been studied in the 10,000 g supernatant fraction of liver homogenates of female rats. Efficient conversion to cholesterol was observed. Other labeled compounds recovered after incubation of [3 alpha-3H]14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol with the enzyme preparations include the unreacted substrate, 5 alpha-cholesta-7,14-dien-3 beta-ol, 5 alpha-cholesta-8,14-dien-3 beta-ol, cholesta-5,7-dien-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, 5 alpha-cholest-8-en-3 beta-ol, and 5 alpha-cholest-7-en-3 beta-ol. In addition, significant amounts of incubated radioactivity were recovered in steryl esters. The steroidal components of these esters were found to contain labeled 14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol, 5 alpha-cholesta-8,14-dien-3 beta-ol, 5 alpha-cholesta-7,14-dien-3 beta-ol, 5 alpha-cholest-8-en-3 beta-ol, 5 alpha-cholest-7-en-3 beta-ol, and cholesterol.     

The stereochemistry of hydrogen elimination during 7,8-double bond formation by Tetrahymena pyriformis

It is shown that formation of the 7,8-double bond in the conversion of cholesterol into cholesta-5,7,22-trien-3beta-ol involves the removal of the 7beta- and 8beta-hydrogen atoms.     

Side chain-hydroxylated sterols of the red alga Asparagopsis armata: Significant products or artifacts due to autoxidation?

Sterols characterized by an allylic hydroxyl group in the side chain, such as stigmasta-5,28-diene-3 beta, 24 epsilon-diol (1), cholesta-5,23-diene-3 beta,25-diol (2) and cholesta-5,25-diene-3 beta,24 epsilon-diol (3), have been identified several times in various marine algae. Their origin was considered as doubtful: they could have been bona fide constituents of the alga, or be artifacts caused by autoxidation during the isolation process. We have shown that the dihydroxy steroids 2 and 3 can indeed be produced by the autoxidation of cholesta-5,24-dien-3 beta-ol (desmosterol) (5), but that they are nevertheless present in the taxonomic significance.     

Inhibitors of ergosterol biosynthesis and growth of the trypanosomatid protozoan Crithidia fasciculata

Six nitrogen-, sulfur- and cyclopropane-containing derivatives of cholestanol were examined as inhibitors of growth and sterol biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The concentrations of inhibitors in the culture medium required for 50% inhibition of growth were 0.32 microM for 24-thia-5 alpha,20 xi-cholestan-3 beta-ol (2), 0.009 microM for 24-methyl-24-aza-5 alpha,20 xi-cholestan-3 beta-ol (3), 0.95 microM for (20,21),(24,-25)-bis-(methylene)-5 alpha,20 xi-cholestan-3 beta-ol (4), 0.13 microM for 22-aza-5 alpha,20 xi-cholestan-3 beta-ol (5), and 0.3 microM for 23-azacholestan-3-ol (7). 23-Thia-5 alpha-cholestan-3 beta-ol (6) had no effect on protozoan growth at concentrations as high as 20 microM. Ergosterol was the major sterol observed in untreated C. fasciculata, but significant amounts of ergost-7-en-3 beta-ol, ergosta-7,24(28)-dien-3 beta-ol, ergosta-5,7,22,24(28)-tetraen-e beta-ol, cholesta-8,24-dien-3 beta-ol, and, in an unusual finding, 14 alpha-methyl-cholesta-8,24-dien-3 beta-ol were also present. When C. fasciculata was cultured in the presence of compounds 2 and 3, ergosterol synthesis was suppressed, and the principal sterol observed was cholesta-5,7,24-trien-3 beta-ol, a sterol which is not observed in untreated cultures. The presence of this trienol strongly suggests that 2 and 3 specifically inhibit the S-adenosylmethionine:sterol C-24 methyltransferase but do not interfere with the normal enzymatic processing of the sterol nucleus. When C. fasciculata was cultured in the presence of compounds 5 and 7, the levels of ergosterol and ergost-7-en-3 beta-ol were suppressed, but the amounts of the presumed immediate precursors of these sterols, ergosta-5,7,22,24(28)-tetraen-3 beta-ol and ergosta-7,24-(28)-dien-3 beta-ol, respectively, were correspondingly increased. These findings suggest that 5 and 7 specifically inhibit the reduction of the delta 24(28) side chain double bond. When C. fasciculata was cultured in the presence of compound 4, ergosterol synthesis was suppressed, but the sterol distribution in these cells was complex and not easily interpreted. Compound 6 had no significant effect on sterol synthesis in C. fasciculata.     

Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes.

The dehydrogenation reaction of cholest-7-en-3beta-ol (I) to cholesta-5,7-dien-3beta-ol (II) in the presence of NADH was studied in rat liver microsomes and in microsomal acetone powder preparations, using [3alpha-3H]cholest-7-en-3beta-ol. It was found that the reaction was inhibited by menadione, adenosine diphosphate, potassium ferricyanide, and cytochrome c while p-cresol had no effect. These results indicated the participation of a microsomal electron transport system in the dehydrogenation of cholest-7-en-3beta-ol. The conversion of cholest-7-en-3beta-ol to cholesta-5,7-dien-3beta-ol was also observed in the absence of NADH when ascorbic acid was included in the incubation mixture. However, the ascorbic acid-catalyzed dehydrogenation was not inhibited by potassium ferricyanide. Immunological evidence that microsomal cytochrome b5 is involved in the dehydrogenation of (I) to (II) was obtained. Antibodies specific for rat liver microsomal cytochrome b5 were elicited in rabbits. The anticytochrome b5 immunoglobulin fraction inhibited rat liver microsomal NADH-cytochrome c reductase but not NADPH-cytochrome c reductase. Also, the extent of reduction of cytochrome b5 was not affected by the antibodies. The conversion of (I) to (II) by rat liver microsomes was inhibited (73%) by anticytochrome b5 immunoglobulin at a ratio of microsomal protein:immunoglobulin of 1:5.6. These results are consistent with the participation of microsomal cytochrome b5 in the introduction of the C-5 double bond in cholesterol biosynthesis. A close analogy of the microsomal dehydrogenation of fatty acids and of cholest-7-en-3beta-ol is apparent and this suggests a possible similarity in the mechanisms of the two reactions.     

Marine sterols. VIII. Isolation and structure of sarcosterol, a new sterol with a delta17(20)-double bond from the soft coral Sarcophyton glaucum.

The sterol mixture of the southern Japan's soft coral, Sarcophyton glaucum, was found to contain 11 sterols including a novel sterol, 23,24 xi-dimethylcholesta-5,22-dien-3 beta-ol and a new diunsaturated C29 sterol. 22,23-Dihydrobrassicasterol and gorgosterol were the major components in free- and esterified sterols respectively. Brassicasterol was found in S. glaucum, in contrast to the ubiquity of 24-epibrassicasterol in the marine invertebrates in the northern districts. The new sterol (sarcosterol) was isolated; its structure as 23 xi, 24 xi-dimethylcholesta-5, 17(20)-trans-dien-3 beta-ol was based on spectra evidence and comparison with cholesta-5, 17(20)-trans-dien-3 beta-ol.     

Inhibition of cholesterol synthesis and cell growth by 24(R,S),25-iminolanosterol and triparanol in cultured rat hepatoma cells

24(R,S),25-Iminolanosterol (IL) and triparanol added to cultures of rat hepatoma cells, H4-II-C3 (H4), interrupt the conversion of lanosterol to cholesterol and, depending on their concentrations, cause the accumulation in the cells of intermediates in the lanosterol to cholesterol conversion. At 45 microM, both substances cause the accumulation of 5 alpha-cholesta-8(9),24-dien-3 beta-ol (zymosterol), and at the low concentration of 4.5 microM, they cause the accumulation of cholesta-5.24-dien-3 beta-ol (desmosterol). The effect of intermediate concentrations of 9 or 22.5 microM of either substance is to cause the accumulation in the cells of three sterols: cholesta-5,7,24-trien-3 beta-ol, zymosterol, and desmosterol. The synthesis of these intermediary sterols, not found normally in H4 cells, is particularly pronounced in cultures kept in lipid-depleted media that contain the inhibitors and proceeds by the use of endogenous substrates at the expense of cholesterol. The synthesis of cholesterol from [14C]acetate or [2-14C]mevalonate is completely blocked by either inhibitor even at 4.5 microM. IL or triparanol inhibits the growth of H4 cells. Cells seeded into either full growth or lipid-depleted medium containing 22.5 microM IL will not grow unless the media are supplemented with low density lipoproteins (60 micrograms/ml). Supplementation of the media with 4.6 mM mevalonate does not counteract the inhibitory effect of IL on cell growth.     

Occurrence of isomeric dehydrocholesterols in human plasma.

Three isomeric dehydrocholesterols were found in plasma from healthy subjects and patients with abnormal production or metabolism of cholesterol. These chemically labile steroids were isolated by a mild liquid-solid extraction procedure using octadecylsilane-bonded silica as sorbent. Sterol-protein interactions were minimized by diluting plasma with aqueous isopropanol. The dehydrocholesterols were identified by high-performance liquid chromatography-ultraviolet spectroscopy and gas chromatography-mass spectrometry as cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), 5 alpha-cholesta-6,8(9)-dien-3 beta-ol (isodehydrocholesterol), and tentatively as cholesta-5,8(9)-dien-3 beta-ol. There was a strong positive correlation between plasma levels of the two former compounds, isodehydrocholesterol levels usually being about 1.4 times higher than those of 7-dehydrocholesterol. The median concentration of 7-dehydrocholesterol in plasma from healthy subjects was 52 ng/ml. Similar concentrations were found in colectomized patients (median concentration 47 ng/ml) and patients with extrahepatic cholestasis and alcoholic liver cirrhosis (median concentrations 79 and 67 ng/ml, respectively). Patients with ileal resection or under treatment with cholestyramine had elevated levels (median concentrations 142 and 160 ng/ml, respectively) whereas patients with primary biliary cirrhosis had subnormal levels (median concentration 26 ng/ml). The results are consistent with a positive correlation between levels of the dehydrocholesterols in plasma and the rate of cholesterol synthesis. The sterols were also analyzed in human skin and bile and the results indicate that the liver may be an important source of isodehydrocholesterol.     

Phospholipid studies of marine organisms: 26. Interactions of some marine sterols with 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC) in model membranes.

The thermotropic behavior of multilamellar vesicles (MLV) composed of different mole fractions of various marine sterols and 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC) was examined by differential scanning calorimetry (DSC), and was compared to pure SOPC as well as their mixtures with cholesterol. The marine sterols investigated were capable of interacting with the phospholipid bilayers. Upon addition of marine sterols, the apparent transition temperature (Tm) of SOPC decreased significantly. Desmosterol (cholesta-5,24-dien-3 beta-ol) had the least interaction with SOPC, as reflected by the larger delta H values of its mixtures with the phospholipid. Fucosterol (24-ethylcholesta-5,24(28)-dien-3 beta-ol) showed a non-linear trend as the mole percent of the sterol increased. Mixtures of sutinasterol (24R-24-ethyl-26,26-dimethylcholesta-7,25(27)-dien-3 beta-ol) with SOPC had similar enthalpy values to cholesterol. The shape of the SOPC/marine sterol endotherm and their delta H values were not identical when liposomes prepared by dialysis were compared to MLV.     

Inhibition of sterol synthesis by delta 5-sterols in a sterol auxotroph of yeast defective in oxidosqualene cyclase and cytochrome P-450

Synthesis of ergosterol is demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the medium sterol was 5 alpha-cholestan-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, or 24 beta-methyl-5 alpha-cholest-8(14)-en-3 beta-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol (24 beta-methylcholesta-5,7,22E-trien-3 beta-ol). Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labeling from [3H]acetate. Except for some cholest-5-en-3 beta-ol (cholesterol) which was derived from the 5 alpha-cholestan-3 beta-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the medium sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5 alpha-cholest-8(9)-en-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol (lathosterol) were converted to cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol. The most attractive of the possible explanations for our observations is the assumption of two genetic compartments for synthesis of sterols, one of which has and one of which has not been affected by the two mutations. The ability, despite the mutations, to synthesize small amounts of ergosterol which could act to regulate the cell cycle may also explain why this mutant can grow aerobically with cholesterol (acting in the bulk membrane role) as the sole exogenous sterol.     

Measurement of the absolute rates of cholesterol biosynthesis in isolated rat liver cells.

Triparanol [2-(4-chlorophenyl)-1-(4-diethylaminoethoxyphenyl)-1-p-tolylethanol] at a concentration of 2 micronm has no effect on the overall conversion of [2=14C]acetate into C27 sterols by isolated liver cells. In the presence of triparanol, however, the formation of radioactive cholesterol is inhibited by 85-90% and the balance of radioactivity appears in the C27 sterol desmosterol (cholesta-5,24-dien-3beta-ol). The very small weights of desmosterol which accumulate under these conditions were, as a routine, quantitatively converted into the heptafluorobutyrate 3-enol ester of cholesta-4,24-dien-3-one. This derivative has a high electron-capturing capability, a property that enables extremely small quantities (less than 0.25pmol) of the material to be accurately measured by gas chromatography with electron-capture detection. Measurements of the mass and specific radioactivity of the newly biosynthesized desmosterol formed in the presence of triparanol provides an accurate assessment of the amount of cholesterol that would be synthesized by the liver cells in the absence of the drug.     

Cyclodextrin-catalyzed extraction of fluorescent sterols from monolayer membranes and small unilamellar vesicles

This study examined the kinetics of sterol desorption from monolayer and small unilamellar vesicle membranes to 2-hydroxypropyl-beta-cyclodextrin. The sterols used include cholesterol, dehydroergosterol (ergosta-5,7,9,(11),22-tetraen-3beta-ol) and cholestatrienol (cholesta-5,7,9,(11)-trien-3beta-ol). Desorption rates of dehydroergosterol and cholestatrienol from pure sterol monolayers were faster (3.3-4.6-fold) than the rate measured for cholesterol. In mixed monolayers (sterol: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 30:70 mol%), both dehydroergosterol and cholestatrienol desorbed faster than cholesterol. clearly indicating a difference in interfacial behavior of these sterols. In vesicle membranes desorption of dehydroergosterol was slower than desorption of cholestatrienol, and both rates were markedly affected by the phospholipid composition. Desorption of sterols was slower from sphingomyelin as compared to phosphatidylcholine vesicles. Desorption of fluorescent sterols was also faster from vesicles prepared by ethanol-injection as compared to extruded vesicles. The results of this study suggest that dehydroergosterol and cholestatrienol differ from cholesterol in their membrane behavior, therefore care should be exercised when experimental data derived with these probes are interpreted.