Sterol metabolism. XXVI. Pyrolysis of some sterol allylic alcohols and hydroperoxides

The thermal decomposition of the allylic alcohols 5α-cholest-6-ene-3β,5-diol, cholest-5-ene-3β,7α-diol, and cholest-5-ene-3β,7β-diol and of the allylic hydroperoxides 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide, 3β-hydroxycho lest-5-ene-7α-hydroperoxide, and 3β-hydroxycholest-5ene-7β-hydroperoxide to six common major pyrolysis products cholest-5-ene-3β,7α-diol, cholest-5-ene-3β,7β-diol, 3β-hydroxycholest-5-en-7-one, cholesta-3,5-dien-7-one, cholesta-4,6-dien-3-one, and cholesta2,4,6-triene was established.     

The synthesis of some cholest-7-en-6-one derivatives related to ecdysones

Cholest-7-en-6-one, 14α-hydroxycholest-7-en-6-one, 2β, 3β,5α-trihydroxycholest-7-en-6-one and ten other related compounds have been synthesized by efficient routes from cholesterol. The named compounds possess some of the characteristics of the insect moulting hormones, ecdysones.     

Sterol metabolism. XLIII. The oxidation of cholest-4-en-3β-ol by singlet molecular oxygen

The photosensitized oxidation of cholest-4-en-3β-ol in which singlet molecular oxygen is implicated yielded cholest-4-en-3-one and the isomeric epoxides 4α,5-epoxy-5α-cholestan-3-one and 4β,5-epoxy-5β-cholestan-3-one, the epoxides being formed in the ratio 3 : 1. Oxidation of cholest-4-en-3-one by alkaline hydrogen peroxide likewise yielded the isomeric 4,5-epoxides but in the ratio 1 : 7.4. Attempted use of cholest-4-en-3β-ol to intercept singlet molecular oxygen putatively generated in the disproportionation of hydrogen peroxide gave a very complex product mixture of over 50 components from which only cholest-4-en-3-one could be identified. However, neither isomeric 4,5-epoxycholestan-3-one was detected among the products. These data establish that it is unwarranted to infer the action of single molecular oxygen in systems containing cholest-4-en-3β-ol merely by product analysis where the product 4α,5-epoxy-5α-cholestan-3-one is formed.     

Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C₇ having a relatively weak carbon–hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen (¹ΔgO₂) and ozone (O₃) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5β-, 6α- and 6β-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ_5,6 –double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.     

Sterol metabolism: XXXVII. On the oxidation of cholesterol by dioxygenases

The oxidation of cholesterol by plant and mammalian dioxygenases yielding cholesterol 7α- and 7β-hydroperoxides has been demonstrated. Cholesterol oxidation is coupled to the oxygenation of polyunsaturated fatty acid esters by soybean lipoxygenase, to the reduction of hydrogen peroxide catalyzed by horseradish peroxidase, and to the oxidation of NADPH by the NADPH-dependent microsomal lipid peroxidation system of rat liver. The initially formed epimeric cholesterol 7-hydroperoxides are transformed in each case to the commonly encountered corresponding 7-alcohol and 7-ketone derivatives. These dioxygenase transformations thus mimic in detail the radiation-induced free radical oxidation of cholesterol by molecular oxygen. Electronically excited (singlet) molecular oxygen is not implicated in these transformations.     

Optical-rotatory-dispersion studies of compounds related to cholesterol in liposomes and the membranes of erythrocyte `ghosts''

1. Steroid molecules containing the alpha,beta-unsaturated oxo group in various positions were incorporated with egg phosphatidylcholine into liposomes and into human erythrocyte membranes. 2. The liposomes formed contained 0.3-0.94mol of steroid/mol of phospholipid and the steroids replaced 19-76% of the erythrocyte membrane sterol. 3. The optical rotatory dispersion (o.r.d.) spectra of the steroids in these structures were compared with those obtained in solvents of different polarity. 4. The o.r.d. spectra of cholesta-4,6-dien-3-one and 3-hydroxycholest-3-en-2-one in liposomes resembled those obtained with polar solvents such as ethanol or triethyl phosphate-water (1:1, v/v). 5. The o.r.d. spectra of 3-hydroxycholest-7-en-6-one and 3-hydroxycholest-5-en-7-one in liposomes resembled those obtained with moderately polar solvents such as dioxan. 6. The o.r.d. spectrum of 3-hydroxycholest-8(14)-en-15-one in liposomes resembled those obtained with non-polar solvents such as cyclohexane. 7. 3-Hydroxycholest-3-en-2-one did not exchange with erythrocyte membrane cholesterol, but the other steroids did do so and the o.r.d. spectra of the membranes containing them closely resembled those obtained with liposomes. 8. From the results, the position of sterol molecules with respect to the phospholipid molecules in liposomes and membranes of human erythrocyte ;ghosts' can be deduced.     

7-Ketocholesterol and cholestane-triol increase expression of SMO and LXRα signaling pathways in a human breast cancer cell line

Oxysterols are 27-carbon oxidation products of cholesterol metabolism. Oxysterols possess several biological actions, including the promotion of cell death. Here, we examined the ability of 7-ketocholesterol (7-KC), cholestane-3β-5α-6β-triol (triol), and a mixture of 5α-cholestane-3β,6β-diol and 5α-cholestane-3β,6α-diol (diol) to promote cell death in a human breast cancer cell line (MDA-MB-231). We determined cell viability, after 24-h incubation with oxysterols. These oxysterols promoted apoptosis. At least part of the observed effects promoted by 7-KC and triol arose from an increase in the expression of the sonic hedgehog pathway mediator, smoothened. However, this increased expression was apparently independent of sonic hedgehog expression, which did not change. Moreover, these oxysterols led to increased expression of LXRα, which is involved in cellular cholesterol efflux, and the ATP-binding cassette transporters, ABCA1 and ABCG1. Diols did not affect these pathways. These results suggested that the sonic hedgehog and LXRα pathways might be involved in the apoptotic process promoted by 7-KC and triol.     

Analysis of native and oxidized low-density lipoprotein oxysterols using gas chromatography—mass spectrometry with selective ion monitoring

SUMMARY

Cholesterol oxidation products have been demonstrated to possess a wide variety of biological properties and have been implicated in playing an important role in the development of atherosclerosis. We have developed an analytical method using capillary gas chromatography-mass spectrometry (GC-MS) for the analysis of cholesterol oxidation products in low-density lipoprotein (LDL). The method uses programmed multiple selected ion monitoring (SIM), providing enhanced sensitivity and accuracy of peak detection over full-scan mass spectra. The major oxidation products of cholesterol in oxidized LDL were identified as 7β-hydroxy-cholesterol and 7-keto-cholesterol. Minor products included 4β-hydroxy-cholesterol, 6β-hydroxy-cholesterol and cholesterol-5α,6α-epoxide. Native LDL contains 7-lathosterol, which is a biosynthetic precursor of cholesterol, as well as low levels of 7β-hydroxy-cholesterol and 7-keto-cholesterol. 7-Lathosterol was not detected in oxidized LDL. A time course oxidation of native LDL with 8 μM CuCl₂ demonstrated a rapid increase in 7β-hydroxy-cholesterol and 7-keto-cholesterol over the first 4 h. Cholesterol—5α,6α-epoxide, and β4-hydroxy- and 6β-hydroxy-cholesterol levels increased gradually, while 7-lathosterol decreased over the same period. This method was used to measure the levels of 7-lathosterol and cholesterol oxides in the LDL of 20 healthy subjects in order to establish the mean concentration and a reference range. This method can be used for the characterization and quantitation of oxysterols in native and oxidized LDL and may afford an additional index of oxidative modification of plasma lipoproteins.     

Biosynthesis of polar steroids from the Far Eastern starfish Patiria (=Asterina) pectinifera. Cholesterol and cholesterol sulfate are converted into polyhydroxylated sterols and monoglycoside asterosaponin P1 in feeding experiments

For the first time, it is experimentally established that the dietary cholesterol and cholesterol sulfate are biosynthetic precursors of polyhydroxysteroids and related low molecular weight glycosides in starfishes. These deuterium labeled precursors were converted into partly deuterated 5α-cholestane-3β,6α,7α,8,15α,16β,26-heptaol, 5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol, and steroid monoside asterosaponin P₁ in result of feeding experiments on the Far Eastern starfish Patiria (=Asterina) pectinifera. The incorporations of deuterium were established by MS and NMR spectroscopy. Scheme of the first stages of biosynthesis of polar steroids in these animals was suggested on the basis of inclusion of three from six deuterium atoms and determination of their positions in biosynthetic products, when [2,2,3,4,4,6-²H₆]cholesterol 3-sulfate was used as precursor. It was also shown that labeled cholesterol is transformed into Δ⁷-cholesterol (lathosterol) in digestive organs and gonads of the starfish.     

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.     

Inhibitors of sterol biosynthesis. Syntheses of 14α-alkyl substituted 15-oxygenated sterols

The chemical syntheses of a number of 14α-alkyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 14α-ethyl-5α-cholest-7-en-3β-ol-15-one, bis-3β,15α-acetoxy-14α-ethyl-5α-cholest-7-ene, 3β-acetoxy-14α-ethyl-5α-cholest-7-en-15β-ol, 14α-ethyl-5α-cholest-7-en-3β,15β-diol, 14α-ethyl-5α-cholest-7-en-3β,15α-diol, 3β-hexadecanoyloxy-14α-ethyl-5α-cholest-7-en-15α-ol, 3β-hexadecanoyloxy-14α-ethyl-5α-cholest-7-en-15β-ol, bis-3β,15α-hexadecanoyloxy-14α-ethyl-5α-cholest-7-ene, 3β-hexadecanoyloxy-14α-ethyl-5α-cholest-7-en-15-one, 3α-benzoyloxy-14α-ethyl-5α-cholest-7-en-15-one, 14α-ethyl-5α-cholest-7-en-3α-ol-15-one, 14α-ethyl-5α-cholest-7-en-15-on-3β-yl pyridinium sulfate, 14α-ethyl-5α-cholest-7-en-15-on-3β-yl potassium sulfate (monohydrate), 14α-ethyl-5α-cholest-7-en-15-on-3α-yl pyridinium sulfate, 14α-ethyl-5α-cholest-7-en-15-on-3α-yl potassium sulfate (monohydrate), 3β-ethoxy-14α-ethyl-5α-cholest-7-en-15-one, 3β-acetoxy-14α-n-propyl-5α-cholest-7-en-15-one, 14α-n-propyl-5α-cholest-7-en-3β-ol-15-one, bis-3β, 15α-acetoxy-14α-n-propyl-5α-cholest-7-ene, 3β-acetoxy-14α-n-propyl-5α-cholest-7-en-15β-ol, 14α-n-propyl-5α-cholest-7-en-3β, 15α-diol, 14α-n-propyl-5α-cholest-7-en-3β, 15β-diol, 14α-n-butyl-5α-cholest-7-en-3β-ol-15-one, 3β-acetoxy-14-α-n-butyl-5α-cholest-7-en-15-one, bis-3β,15α-acetoxy-14α-n-butyl-5α-cholest-7-ene, 3β-acetoxy-14α-n-butyl-5α-cholest-7-en-15β-ol, 14α-n-butyl-5β-cholest-7-en-3β, 15β-diol, and 14α-n-butyl-5α-cholest-7-en-3β, 15α-diol.     

Stereoselectivity of raney nickel catalyst in hydrogenolysis of a steroidal α,β-unsaturated epoxide. Chemical synthesis of 3β-benzoyloxy-5α-cholest-8(14)-en-15α-ol

Hydrogenation of 3β-benzoyloxy-14α, 15α-epoxy-5α-cholest-7-ene in benzene over a Raney nickel catalyst gave 3β-benzoyloxy-5α-cholest-8(14)-en-15α-ol and 3β-benzoyloxy-5α-cholest-8(14)-ene in 39% and 46% yields, respectively. Hydrogenation of the same α,β-unsaturated epoxy steryl ester under the same conditions except with the inclusion of triethylamine (4%) gave 3β-benzoyloxy-5α-cholest-8(14)-en-15α-ol in 89% yield.     

Concerning the structure of 7α,15β-dichloro-5α-cholest-8(14)-en-3β-ol,a novel inhibitor of cholesterol biosynthesis

Treatment of 3β-p-bromobenzoyloxy-14α, 15α-epoxy-5α-cholest-7-ene with gaseous HCI in chloroform at ?25°C gave 3β-p-bromobenzoyloxy-7α, 15β-dichloro-5α-cholest-8(14)-ene in 93% yield. The structure of the latter compound was unequivocally established by the results of X-ray crystallographic analysis.     

Enzymatic isomerization (Δ7 → Δ8) of the nuclear double bond of 14α-alkyl substituted sterol precursord of cholesterol

The catalysis by rat liver microsomes under anaerobic conditions, of the conversion of [3α-³H]14α-methyl-5α-cholest-7-en-3β-ol and of [2,4-³H]14α-hydroxymethyl-5α-cholest-7-en-3β-ol to labeled 14α-methyl-5α-cholest-8-en-3β-ol and 14α-hydroxymethyl-5α-cholest-8-en-3β-ol, respectively, has been demonstrated. This finding is of importance in evaluating past research in this area and in consideration of pathways and mechanisms involved in enzymatic removal of carbon atom 32 of 14α-methyl sterols. Also described herein are syntheses of [2,4-³H]14α-hydroxymethyl-5α-cholest-7-en-3β-ol and 3β-acetoxy-14α-methyl-5α-cholest-8-ene.     

The epimeric 20-hydroperoxy-5-pregnen-3 -ols: thermal and enzymatic decomposition

The isolation of 20α-hydroperoxy-5-pregnen-3β-ol and its 20β-isomer from air aged cholesterol is described. The structures of these new steroids are deducted from their physicochemical properties and confirmed by borohydride reduction to the known epimeric 5-pregnene-3β, 20-diols. Formation of the 20α-hydroperoxy-5-pregnen-3β-ol during the autoxidation process is suggested to result from the interaction of molecular oxygen with a 3β-hydroxy-5-pregnen-20α-yl radical, a specie which may be formed upon decomposition of the 25-hydroperoxy-5-cholesten-3β-ol. Formation of the 20β-hydroperoxy-epimer is shown to result partially from isomerization of the 20α-hydroperoxy-5-pregnen-3β-ol. Thermal decomposition of both isomers gives pregnenolone (3β-hydroxy-5-pregnen-20-one) as the major product together with the corresponding 5-pregnene-3β, 20-diol, 5-androsten-3β-ol and a small amount of 5-androstene-3β, 17β-diol and 5, 16-androstadien-3β-ol. Incubation of either hydroperoxide with adrenocortex microsomal and mitochondrial preparations gave pregnenolone and the corresponding steroid alcohol as the sole products. These results are discussed in comparison with the earlier reported studies on the 20α-hydroperoxy-5-cholesten-3β-ol and in terms of the possible role of steroid hydroperoxides as transit species in the biogenesis of steroid hormones.     

Biotransformation of 7α-hydroxy- and 7-oxo-ent-atis-16-ene derivatives by the fungus Gibberella fujikuroi

The microbiological transformation of 7α,19-dihydroxy-ent-atis-16-ene by the fungus Gibberella fujikuroi gave 19-hydroxy-7-oxo-ent-atis-16-ene, 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene, 7α,11β,19-trihydroxy-ent-atis-16-ene and 7α,16β,19-trihydroxy-ent-atis-16-ene, while the incubation of 19-hydroxy-7-oxo-ent-atis-16-ene afforded 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene and 16β,17-dihydroxy-7-oxo-ent-atisan-19-al. The biotransformation of 7-oxo-ent-atis-16-en-19-oic acid gave 6β-hydroxy-7-oxo-ent-atis-16-en-19-oic acid, 6β,16β,17-trihydroxy-7-oxo-19-nor-ent-atis-4(18)-ene and 3β,7α-dihydroxy-6-oxo-ent-atis-16-en-19-oic acid.     

Cytotoxic terpenoids from Juglans sinensis leaves and twigs

Bioassay-guided fractionation of an 80% MeOH extract of Juglan sinensis leaves and twigs has resulted in the isolation of three new triterpenes (1-3) and two new sesquiterpenes (4-5) along with two known sesquiterpenes (6-7). The new compounds were determined to be 3β, 11α, 19α, 24, 30-pentahydroxy-20β, 28-epoxy-28β-methoxy-ursane (1), 1α, 3β-dihydroxy-olean-18-ene (2), 2α, 3α, 23-trihydroxy-urs-12-en-28-oic acid 28-O-β-d-glucopyranoside (3), (4S, 5S, 7R, 8R, 14R)-8, 11-dihydroxy-2, 4-cyclo-eudesmane (4), 15-hydroxy-α-eudesmol-11-O-β-d-glucopyranoside (5), by spectroscopic analysis. The cytotoxicity of compounds (1-7) against four cancer cell lines such as B16F10, Hep-2, MCF-7 and U87-MG was evaluated. Compounds 1, 2, 6 and 7 showed potent cytotoxicity against all of four cancer cell lines, respectively.     

Sterol synthesis. Synthesis of 15-oxygenated 5α, 14β-cholest-7-en-3β-ol derivatives

Treatment of 3β-benzoyloxy-14α, 15α-epoxy-5α-cholest-7-ene with boron trifluoride-etherate gave, in 43% yield, 3β-benzoyloxy-5α, 14β-cholest-7-en-15-one with the unnatural C ring juncture. Reduction of the latter compound with lithium aluminum hydride gave 15α, 14β-cholest-7-en-3β, 15α-diol and 5α, 14β-cholest-7-en-3β, 15β-diol in 9% and 81% yields, respectively.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Synthesis and cytotoxic analysis of some disodium 3β,6β-dihydroxysterol disulfates

Disodium 3β,6β-dihydroxy-5α-cholestane disulfate (1) was synthesized in 4 steps with a high overall yield from cholesterol. First, cholesterol (4a) was converted to cholest-4-en-3,6-dione (5a) via oxidation with pyridinium chlorochromate (PCC) and then 5a was reduced by NaBH₄ in the presence of NiCl₂ to produce cholest-3β,6β-diol (6a). The reaction of 6a with the triethylamine-sulfur trioxide complex generated diammonium 3β,6β-dihydroxy-5α-cholestane disulfate (7a) and the treatment of 7a by cation exchange resin 732 (sodium form)(Na⁺) yielded the target steroid 1. Disodium 24-ethyl-3β,6β-dihydroxycholest-22-ene disulfate (2) and disodium 24-ethyl-3β,6β-dihydroxycholestane disulfate (3) were synthesized using a similar method. The cytotoxicity of these compounds against Sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells was investigated. Our results indicate that presence of a cholesterol-type side chain at position 17 is necessary for their biological activity.