28-homobrassinolide regulates antioxidant enzyme activities and gene expression in response to salt- and temperature-induced oxidative stress in Brassica juncea.

Brassinosteroids (BRs) are a group of naturally occurring plant steroid hormones that can induce plant tolerance to various plant stresses by regulating ROS production in cells, but the underlying mechanisms of this scavenging activity by BRs are not well understood. This study investigated the effects of 28-homobrassinolide (28-HBL) seed priming on Brassica juncea seedlings subjected to the combined stress of extreme temperatures (low, 4 °C or high, 44 °C) and salinity (180 mM), either alone or supplemented with 28-HBL treatments (0, 10-6, 10-9, 10-12 M). The combined temperature and salt stress treatments significantly reduced shoot and root lengths, but these improved when supplemented with 28-HBL although the response was dose-dependent. The combined stress alone significantly increased H2O2 content, but was inhibited when supplemented with 28-HBL. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) increased in response to 28-HBL. Overall, the 28-HBL seed priming treatment improved the plant's potential to combat the toxic effects imposed by the combined temperature and salt stress by tightly regulating the accumulation of ROS, which was reflected in the improved redox state of antioxidants.

Functional characterization of CYP71D443, a cytochrome P450 catalyzing C-22 hydroxylation in the 20-hydroxyecdysone biosynthesis of Ajuga hairy roots.

20-Hydroxyecdysone (20HE), a molting hormone of insects, is also distributed among a variety of plant families. 20HE is thought to play a role in protecting plants from insect herbivores. In insects, biosynthesis of 20HE from cholesterol proceeds via 7-dehydrocholesterol and 3ß,14α-dihydroxy-5ß-cholest-7-en-6-one (5ß-ketodiol), the latter being converted to 20HE through sequential hydroxylation catalyzed by four P450 enzymes, which have been cloned and identified. In contrast, little is known about plant 20HE biosynthesis, and no biosynthetic 20HE gene has been reported thus far. We recently proposed involvement of 3ß-hydroxy-5ß-cholestan-6-one (5ß-ketone) in 20HE biosynthesis in the hairy roots of Ajuga reptans var. atropurpurea (Lamiaceae). In this study, an Ajuga EST library was generated from the hairy roots and P450 genes were deduced from the library. Five genes with a high expression level (CYP71D443, CYP76AH19, CYP76AH20, CYP76AH21 and CYP716D27) were screened for a possible involvement in 20HE biosynthesis. As a result, CYP71D443 was shown to have C-22 hydroxylation activity for the 5ß-ketone substrate using a yeast expression system. The hydroxylated product, 22-hydroxy-5ß-ketone, had a 22R configuration in agreement with that of 20HE. Furthermore, labeling experiments indicated that (22R)-22-hydroxy-5ß-ketone was converted to 20HE in Ajuga hairy roots. Based on the present results, a possible 20HE biosynthetic pathway in Ajuga plants involved CYP71D443 is proposed.

Biosynthesis of 20-hydroxyecdysone in plants: 3ß-hydroxy-5ß-cholestan-6-one as an intermediate immediately after cholesterol in Ajuga hairy roots.

3ß-Hydroxy-5ß-cholestan-6-one was identified in the EtOAc extract of Ajuga hairy roots by micro-analysis using LC-MS/MS in the multiple reaction mode (MRM). Furthermore, administration of (2,2,4,4,7,7-(2)H6)- and (2,2,4,4,6,7,7-(2)H7)-cholesterols to the hairy roots followed by LC-MS/MS analysis of the EtOAc extract of the hairy roots indicated that cholesterol was converted to the 5ß-ketone with hydrogen migration from the C-6 to the C-5 position. These findings, in conjunction with the previous observation that the ketone was efficiently converted to 20-hydroxyecdysone, strongly suggest that the 5ß-ketone is an intermediate immediately formed after cholesterol during 20-hydroxyecdysone biosynthesis in Ajuga sp. In addition, the mechanism of the 5ß-ketone formation from cholesterol is discussed.

Biosynthetic relationship between C28-brassinosteroids and C29-brassinosteroids in rice (Oryza sativa) seedlings.

A crude enzyme solution was prepared from young rice seedlings, and the metabolism of C29-brassinosteroids identified from the seedlings was examined. When 28-homoteasterone was added as a substrate, 28-homotyphasterol, teasterone, and 26-nor-28-homoteasterone were characterized as enzyme products by GC-MS/SIM analysis. With 28-homotyphasterol, 28-homoteasterone, typhasterol, 28-homocastasterone, and 26-nor-28-homotyphasterol were formed and identified as products. When 28-homocastasterone was used, castasterone and 26-nor-28-homocastasterone were identified as products. Together with the reduced biological activity of C29-brassinosteroids and their metabolites in the rice lamina inclination assay, these metabolic studies suggest a biosynthetic sequence, 28-homoteasterone↔28-homotyphasterol→28-homocastasterone for C29-brassinosteroid biosynthesis is connected to the biosynthetic sequence teasterone↔typhasterol→castasterone for C28-brassinosteroids by C-28 demethylation, i.e., in order to increase biological activity in the rice plant. Additionally, the C29-brassinosteroids seem to bio-degrade their C-26 demethylated C28-brassinosteroid analogs to reduce brassinosteroid activity in planta. In conclusion, the biosynthesis of C29-brassinosteroids is a likely alternative route to the biologically-active brassinosteroid, castasterone, in rice.

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 ((1)ΔgO2) 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-, 5ß-, 6α- and 6ß-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 Δ5,6 -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 3ß-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.

Conversion of 3-oxo steroids into ecdysteroids triggers molting and expression of 20E-inducible genes in Drosophila melanogaster.

Ecdysteroids, steroid hormones in insects, coordinate major developmental transitions. During postembryonic development, ecdysone is biosynthesized from dietary cholesterol in the prothoracic gland (PG). Despite extensive studies, the initial conversion process, the so-called "Black Box", has not been characterized. A cytochrome P450 enzyme, Spookier (Spok), is speculated as a rate limiting enzyme in the Black Box during larval-pupal transitions in Drosophila melanogaster. RNAi mediated knockdown of spok expression in the PG results in arrest of molting. Because the developmental arrest can be rescued by application of an appropriate intermediate, we examined potential activities of candidate intermediates in the RNAi-treated larvae. We found that two 3-oxo steroids, cholesta-4,7-diene-3,6-dione-14α-ol (Δ(4)-diketol) and 5ß [H]cholesta-7-ene-3,6-dione-14α-ol (diketol), triggered molting of the RNAi-treated larvae. We also detected an enhancement of the amounts of ecdysteroids in the RNAi-treated larvae by feeding the Δ(4)-diketol or diketol, indicating that the dietary 3-oxo steroids were incorporated and converted into ecdysteroids in vivo. Furthermore, 20-hydroxyecdysone inducible genes were induced in the RNAi-treated larvae by feeding the Δ(4)-diketol or diketol. These results indicate that Δ(4)-diketol and diketol are components of the ecdysteroid biosynthetic pathway and lie downstream of a step catalyzed by Spok.

Effect of 28-homobrassinolide on antioxidant defence system in Raphanus sativus L. under chromium toxicity.

Heavy metals have emerged as major environmental contaminants due to rapid industrialization and urbanization. The genotoxic, mutagenic and carcinogenic effects of heavy metal like chromium (Cr) on man, animals and plants have been documented. In plants, accumulation of heavy metals beyond critical levels generates oxidative stress. This stress is generally overcome by antioxidant defence system and stress shielding phytohormones. Thus, the present study has been focused to analyze the effect of one of imperative group of plant hormones, i.e., brassinosteroids (BRs) which have been reported for its protective properties for wide array of environmental stresses. Raphanus sativus L. (Pusa Chetaki) seeds pre-treated with different concentrations of 28-homobrassinolide (28-HBL) were raised under various concentrations of Cr(VI). It was observed that 28-HBL treatment considerably reduced the impact of Cr-stress on seedlings which was evinced upon analysis of morphological and biochemical parameters of 7-days old radish seedlings. The toxic effects of Cr in terms of reduced growth, lowered contents of chlorophyll (Chl), protein, proline; increased malondialdehyde (MDA) content and elevated metal uptake were ameliorated by applications of 28-HBL. Also, the activities of all the antioxidant enzymes except guaiacol peroxidase (POD), increased significantly when subjected to Cr stress in combination with 28-HBL. Overall, seed pre-soaking treatment of 28-HBL at 10(-7) M was most effective in ameliorating Cr stress. The present work emphasizes the protective role of 28-HBL on regulation of antioxidant enzymes and its possible link in amelioration of stress in plants.

Formation of cholesterol ozonolysis products in vitro and in vivo through a myeloperoxidase-dependent pathway.

3ß-Hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its aldolization product 3ß-hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde (secosterol-B) were recently detected in human atherosclerotic tissues and brain specimens, and they may play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. However, as their origin remains unidentified, we examined the formation mechanism, the stability, and the fate of secosterols in vitro and in vivo. About 40% of secosterol-A remained unchanged after 3 h incubation in the FBS-free medium, whereas 20% and 40% were converted to its aldehyde-oxidation product, 3ß-hydroxy-5-oxo-secocholestan-6-oic acid, and secosterol-B, respectively. In the presence of FBS, almost all secosterol-A was converted immediately to these compounds. Secosterol-B in the medium, with and without FBS, was relatively stable, but ∼30% was converted to its aldehyde-oxidation product, 3ß-hydroxy-5ß-hydroxy-B-norcholestane-6-oic acid (secoB-COOH). When neutrophil-like differentiated human leukemia HL-60 (nHL-60) cells activated with PMA were cultured in the FBS-free medium containing cholesterol, significantly increased levels of secosterol-A and its aldehyde-oxidation product, but not secosterol-B, were formed. This secosterol-A formation was decreased in the culture of PMA-activated nHL-60 cells containing several reactive oxygen species (ROS) inhibitors and scavengers or in the culture of PMA-activated neutrophils isolated from myeloperoxidase (MPO)-deficient mice. Our results demonstrate that secoterol-A is formed by an ozone-like oxidant generated with PMA-activated neutrophils through the MPO-dependent mechanism.

Protective response of 28-homobrassinolide in cultivars of Triticum aestivum with different levels of nickel.

Seeds of five wheat (Triticum aestivum) cultivars (PBW-373, UP-2338, DL-LOK-01, DL-373, and HD-2338) were sown in earthen pots and 10-day-old seedlings were exposed to 0, 50, and 100 µM of nickel (Ni) in the form of nickel chloride. At the 20-day stage, seedlings were sprayed with 0.01 µM of 28-homobrassinolide (HBL). The results of the experiment at the 30-day stage revealed a decline in the dry mass per plant, leaf area, leaf water potential, and net photosynthetic rate with concomitant decline in the activities of various enzymes (viz. carbonic anhydrase and nitrate reductase) with an increasing concentration of Ni. However, an increase in proline content and the activities of catalase, peroxidase, and superoxide dismutase was observed as a result of an increase in Ni concentration. Moreover, the treatment of these stressed plants with HBL enhanced the activities of carbonic anhydrase and nitrate reductase, catalase, peroxidase, and superoxide dismutase. The proline content in the leaves also increased, which is known to act as an osmolyte and reactive oxygen species scavenger. The toxic effects generated by Ni were ameliorated by HBL through an improved antioxidant system and osmolyte. Moreover, improvement of photosynthetic parameters and growth characteristics further strengthen our belief that HBL acted as a potent stress alleviator.

Cholesterol secoaldehyde induces apoptosis in J774 macrophages via mitochondrial pathway but not involving reactive oxygen species as mediators.

Cholesterol secoaldehyde (3beta-hydroxy-5-oxo-5,6-secocholestan-6-al or ChSeco) is an oxysterol known to be formed in reactions of ozone with cholesterol and also when cholesterol-5alpha-hydroperoxide undergoes Hock cleavage. In view of its widespread occurrence and atherogenic potential, we examined the effects of ChSeco on mouse J774 macrophage viability and events associated with apoptosis. A dose-dependent decrease in cell viability, disruptions in mitochondrial transmembrane potential (64+/-5.5%; mean+/-SD, n=3), increased levels of cytosolic cytochrome c (8.8+/-0.84 ng/ml; mean+/-SD, n=3), activation of caspase-3 (ca. 3.6-fold) and caspase-9 (ca.1.8-fold), and increased DNA fragmentation (ca. 5-fold), all indicative of apoptosis, were observed in response to exposure to ChSeco. The apoptotic nature of cell death in macrophages was confirmed by dual staining with acridine orange and ethidium bromide. However, unlike the case with cardiomyoblasts and neuronal cells, the apoptotic process in these immune cells was not mediated by increased levels of reactive oxygen species as indicated by a minimal or no increase in 2',7'-dichlorofluorescein fluorescence. It is suggested that the apoptotic process is mediated via the mitochondrial pathway and that ChSeco formed in biological environments contributes to the initiation, progression, and culmination of atherosclerotic plaque formation, as these processes are critically dependent on macrophage apoptosis.

Lipophilic compounds from the femoral gland secretions of male Hungarian green lizards, Lacerta viridis.

In spite of the importance of chemical signals (pheromones) in the reproductive behaviour of lizards, only a few studies have examined the role of specific chemical compounds as sexual signals. The secreted chemicals vary widely between species but whether this variation reflects phylogenetic or environmental differences remains unclear. Based on mass spectra, obtained by GC-MS, we found 40 lipophilic compounds in femoral gland secretions of male green lizards (Lacerta viridis), including several steroids, alpha-tocopherol, and esters of n-C16 to n-C20 carboxylic acids, and minor components such as alcohols between C12 and C20, squalene, three lactones and one ketone. We compared these chemicals with those previously found in other closely related green lizard species, and discussed how phylogenetical differences and/or environmental conditions could be responsible for the differential presence of chemicals in different lizard species.

Intracellular oxidative stress and cytotoxicity in rat primary cortical neurons exposed to cholesterol secoaldehyde.

Cholesterol secoaldehyde (ChSeco or 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al) has been shown to induce Abeta aggregation and apoptosis in GT1-7 hypothalamic neurons. The present study was undertaken to evaluate the effects of ChSeco on rat primary cortical neuronal cells. ChSeco was cytotoxic at concentrations ranging from 5 to 20 microM, while cholesterol of comparable concentrations showed little or no toxicity. In ChSeco-exposed neuronal cells, there was an increased formation of intracellular peroxide or peroxide-like substance(s), the levels of which were comparable to those found in typical menadione exposures. There was a loss in the mitochondrial transmembrane potential, the extent of which was dependent on concentration of ChSeco employed. Pre-treatment with N-acetyl-L-cysteine (5 mM; 1 h) offered protection against the cytotoxicity and the generation of intracellular oxidants. Cytotoxicity of ChSeco was evidenced by the loss of axonal branches and also condensed apoptotic nuclei in these cells. Immunohistochemical analysis revealed a decreased intracellular Abeta42 staining proportional to the loss in the axonal out growth and dendritic branches. The observed decrease in Abeta42 has been suggested to be due to loss of integrity of dendrites and the plasma membrane, possibly resulting from increased production of reactive oxygen species.

Cholesterol secoaldehyde, an ozonation product of cholesterol, induces amyloid aggregation and apoptosis in murine GT1-7 hypothalamic neurons.

Aldehydic products from ozonation of cholesterol and peroxidation of phospholipids have been shown to accelerate aggregation of amyloid-beta (Abeta) in vitro. Here, we show that 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco), an ozonation product of cholesterol, induces Abeta aggregation, generation of reactive oxygen species (ROS), and cytotoxicity in murine GT1-7 hypothalamic neurons. The formation of Abeta aggregates in situ was dose-dependent at ChSeco concentrations ranging from 1 to 20 microM. The increase in insoluble Abeta aggregates at increasing concentrations of ChSeco was accompanied by a decrease in soluble Abeta as evidenced by Western blot analysis. The formation of ROS in neuronal cells was found to be dose- and time-dependent with the magnitude being higher at 20 microM compared to 10 microM ChSeco or untreated controls. The increase in ROS was associated with depletion of GSH. The cytotoxicity induced by ChSeco involved changes in phosphatidylserine translocation, DNA fragmentation, and caspase 3/7 activity that are characteristic of apoptosis. Pretreatment of neuronal cells with Trolox, a water-soluble analog of alpha-tocopherol offered partial, but significant protection against ChSeco-induced cell death, whereas, N-acetyl-L-cysteine (NAC) completely prevented the cytotoxic effects of ChSeco. NAC and Trolox were without any effects on ChSeco-induced Abeta aggregation. Fibrillogenesis inhibitors, which inhibited Abeta aggregation, did not inhibit cell death induced by ChSeco, implying that ROS generation, and not Abeta aggregation, plays a major role in the observed cytotoxicity. However, since Alzheimer's and other neurodegenerative diseases are slow and progressive, the formation of Abeta aggregates in vivo by ChSeco may have long-term pathological consequences.

A product of ozonolysis of cholesterol alters the biophysical properties of phosphatidylethanolamine membranes.

There is evidence that some products of the reaction of ozone with cholesterol contribute to atherosclerosis. One of these compounds is 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al. We have synthesized this compound and have demonstrated that it reacts with phosphatidylethanolamine to form a Schiff base. The 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al also affects the physical properties of phosphatidylethanolamines. We show by both DSC and X-ray diffraction that it increases the negative curvature of the membrane. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al causes the lamellar phase to become disorganized, resulting in the loss of lamellar periodicity. The chemical and physical interactions of 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al with phosphatidylethanolamines may contribute to damaging effects of this lipid on cell membranes, resulting in pathology.

1,5-Hydride shift in Wolff-Kishner reduction of (20R)-3beta,20, 26-trihydroxy-27-norcholest-5-en-22-one: synthetic, quantum chemical, and NMR studies.

Heating (20R)-3beta,20,26-trihydroxy-27-norcholest-5-en-22-one (1) with hydrazine and KOH at 160 degrees C completely converted the steroid to a diastereoisomeric mixture of the new (20R,22RS)-27-norcholest-5-ene-3beta,20,22-triols (2). Exclusive formation of 2 suggests that the expected Wolff-Kishner reduction to a methylene group at the C-22 ketone in 1 was diverted to the C-26 position by a 1,5-hydride shift. All attempts under acid conditions failed to produce a C-22 phenyl hydrazone from 1. However, reaction of 1 was reacted with phenylhydrazine in hot KOH, gave the C-26 phenylhydrazone 4 as the sole product. Evidently, under alkaline conditions, first a hydride ion undergoes an intramolecular transfer from the C-26 CH(2)OH group to the C-22 ketone in 1, and then the phenylhydrazine traps the newly formed aldehyde. To examine this hypothesis, we constructed computer-simulated transition state models from quantum chemical calculations and then compared data from these models with NMR measurements of the reaction mixtures containing 2. The NMR data showed that the C-22 diastereoisomers of 2 are formed in a nearly 1:1 ratio exactly as predicted from the energy-optimized transition states, which were calculated for intramolecular 1,5-hydride shifts that produced each of the two C-22 diastereoisomers. Accordingly, these results support the hypothesis that an intramolecular 1,5-hydride shift mechanism promotes complete conversion of 1 to 2 under classical Wolff-Kishner reduction conditions.

Oxygenated cholesterols as ligands for cytosolic-nuclear tumor promoter binding protein: yakkasteroids.

Several oxygenated cholesterols and their derivatives (yakkasteroids) were prepared as candidate ligands for cytosolic-nuclear tumor promoter binding protein (CN-TPBP). Among the compounds, a possible natural substance, 3 beta,5 alpha-dihydroxycholestan-6-one (yakkasterone), showed the highest binding affinity to CN-TPBP. It also showed high specificity for binding to CN-TPBP: it does not bind to protein kinase C. Investigation of the structure-activity relationships of yakkasteroids revealed that the structure of the side chain at the 20-position is important for the binding activity to CN-TPBP.

Microbial degradation of 2 alpha, 3 alpha-dihydroxy-5 alpha-cholestan-6-one by Mycobacterium vaccae.

2 alpha,3 alpha-Dihydroxy-5 alpha-cholestan-6-one (3), which had the substitution pattern of brassinosteroids in the A/B-ring moiety, was transformed by Mycobacterium vaccae to give 2 alpha,3 alpha,6 alpha-trihydroxy-5 alpha-androstan-17-one (4) and 2 alpha-hydroxyandrost-4-ene-3,17-dione (5). The structures of these compounds were determined by spectroscopic methods, especially 1H nuclear magnetic resonance studies.

A convenient isomerization of 6-oxo-3 alpha,5-cyclo-5 alpha-steroids to 6-oxo-delta 2-5 alpha-steroids.

Direct isomerization of 6-oxo-3 alpha,5-cyclo-5 alpha-steroids to 6-oxo-delta 2-5 alpha-steroids was accomplished by pyridinium hydrobromide in dimethyl-formamide.

Side-chain oxidation of monooxygenated C27- and C29-steroids in rat liver mitochondria and 18000 x g supernatant.

The 24-, 25- and 26-hydroxylation of 4-cholesten-3 alpha-ol, 4-cholesten-3 beta-ol, 5-cholesten-3 alpha-ol, 5-cholesten-3 beta-ol, 5 alpha-cholestan-3 alpha-ol, 5 alpha-cholestan-3 beta-ol, 5 beta-cholestan-3 alpha-ol, 5 beta-cholestan-3 beta-ol, 4-cholesten-3-one, 5 alpha-cholestan-3-one, 5 beta-cholestan-3-one and the 24 alpha-ethyl derivatives of 5 alpha-cholestan-3 beta-ol, 5 beta-cholestan-3 alpha-ol, 5 beta-cholestan-3 beta-ol and 4-cholestan-3-one were studied in rat liver mitochondria (8500 x g sediment fractions fortified with isocitrate) and in rat liver microsomes (18000 x g supernatants supplemented with NADPH). In the mitochondria, all C27-substrates and probably all C29-substrates were found to be omega-hydroxylated. From 24 alpha-ethyl-5 alpha-cholestan-3 beta-ol and 24 alpha-ethyl-4-cholesten-3-one two omega-hydroxylated products were identified. All C27- but no C29-steroids were found to be 24- and 25-hydroxylated. The yield of omega-hydroxylated metabolites were much higher than those of the 24-, and 25-hydroxylated products. The omega-hydroxylation of the C29-steroids amounted to 3-50% of that found for the corresponding C27-steroids. In the 18000 x g supernatant only one substrate, 5 beta-cholestan-3 alpha-ol, was found to be 25- and 26-hydroxylated and no 24-hydroxylation of any steroid could be detected.

Metabolism of bile alcohols, 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol and 3 alpha,7 alpha,12 alpha-trihydroxy-26,27-dinor-5 beta-cholestan-24-one, in rats.

24-Nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol and 3 alpha,7 alpha,12 alpha-trihydroxy-26,27-dinor-5 beta-cholestan-24-one were administered intraperitoneally to bile fistula rats, and the metabolites excreted in the bile were analyzed. No formation of bile acids from these bile alcohols was observed. 7 alpha,12 alpha,25-Trihydroxy-24-nor-5 beta-cholestane-3 alpha-O-(beta-D-glucopyranosid)uronic acid was identified as the only biliary metabolite of the 24-nor-5 beta-cholestanetetrol. The major metabolite of the trihydroxy-26,27-dinor-5 beta-cholestanone was 7 alpha,12 alpha-dihydroxy-24-oxo-26,27-dinor-5 beta-cholestane-3 alpha-O-(beta-D-glucopyranosid)uronic acid, and the minor metabolite was the glucurono conjugate of 26,27-dinor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 beta-tetrol. The results indicated that in rat liver these C25- and C26-bile alcohols, in contrast to C27-bile alcohols, were not converted into bile acids, and that the glucuronide production became necessary for hepatic elimination of the accumulated bile alcohols.