5-Cholestene-30,26-diol

On a dark background cholesterol (Eluent A, h/ f 20-25) emitted blue, coprostanol (Eluent A, h/ f 25-30) blue, 4-cholesten-3-one (Eluent A, h/ f 40-45) blue, 5a-cholestan-3-one (Eluent A, h/ f 60) blue, coprostanone (Eluent A, h/ f 70) blue, estriol 3-sulfate (Eluent B, h/ f 5-10) yellow, 11-ketoetiocholanolone (Eluent B, h/ f 15-20) blue, estrone (Eluent B, h/ f 20-25) ochre, 11-desoxycorticosterone (Eluent B, h/ f 30-35) yellow, 17a-ethinyl-5-androstene-3p,17p-diol (Eluent B, hRj 45-50) ochre, 4-cholesten-3-one (Eluent B, h/ f 55-60) faint blue and coprostanone (Eluent B, h/ f 65-70) violet fluorescences. 

Fig. 2 Fluorescence scan of a chromatogram track with 100 ng each of estriol-3-sulfate (1), 11-ketoetiocholanone (2), estrone (3) 11-desoxycorticosterone (4) and 17a-ethinyl-5-androsten-3P,17P-diol (5), together with 1 pg each of 4-cholesten-3-one (6) and coprostanone (7) per chromatogram zone.

Weakly fluorescent zones were visible under long-wavelength UV light (X = 365 i (Fig. 1). Cortisone (h/ f 0-5), dienestrol (h/ f 10-15), 4-androstene-3,17-dione (It 50-55) and 4-cholesten-3-one (h/ f 60-65) had an ochre fluorescence. Diethylsti estrol (h/ f 10-15), 17a-ethinyl-l,3,5-estratriene-3,17B-diol (h/ f 25-30) and estro (h/ f 35-40) had a blue emission. 

Fig. 1 Fluorescence scan of a chromatogram track with 1 ng cortisone (1), 100 ng dienestrol (2X 300 ng 17a-ethinyl-l,3,5-estratriene-3,17B-diol (3), 100 ng estrone (4) and 1 ug each of 4-androstene-3,17-dione (5) and 4-cholesten-3-one (6) A. before immersion in Triton X-100, B. after immersion followed by brief drying, C after heating to 120 °C for 10 minutes and D. for a further 20 minutes to increase the fluorescence.

Formation and /S-fission of bicyclic tertiary alkoxyl radicals from the corresponding alcohols are well known [38] [40]. The treatment of 5a-cholestane-3/3,5-diol-3-acetate, VII/70, and the 5/3-alcohol, VII/71, respectively (Scheme VII/15), with one molar equivalent of lead tetraacetate in the presence of anhydrous calcium carbonate gives radical fragmentation reactions. The products are the two (E)- and (Z)-3/3-acetoxy-5,10-seco-l(10)-cholesten-5-ones (VII/72 + VII/73) [40]. The ratio of VII/73 VII/72 is 63 10 [41] [42] [43]. 

One milligram of microsomal protein is added to 0.1 M potassium phosphate buffer (pH 7.4) containing 50 mM NaF, 10 mM dithiothreitol, 1 mM EDTA, 20% glycerol (v/v), 150 iM 5-cholestene-3/3, 7a-diol, and 0.915% CHAPS. The reaction is initiated by 1 mM NAD+ to give a final reaction volume of 1.0 mL. After incubation at 37°C for 5 minutes, the reaction is terminated by adding 2 mL of 95% ethanol. An internal recovery standard, 4-cholesten-3-one (3 fig in methanol) is also added. The steroid products are extracted into 5 mL of petroleum ether (repeated twice). After the ether has been removed at 40°C under a stream of nitrogen, the products are dissolved in 100 fxL of mobile phase and 20 ju.L is injected into the column. The amount of product formed is linear with protein (to 1.5 mg) and with time (up to 10 min, 1 mg protein). The assay is much more sensitive than the direct spectrophotometric assay, and it avoids the use of thin-layer chromatography and radioisotopes described in other methods. 

A preliminary report" states that reaction of cholesteryl benzoate with (-butyl hydroperoxide and a catalytic amount of cuprous bromide in benzene gave equal parts of A -cholestene-3, 7a- and -3, 7j8-diol dibenzoate. Here allylic attack is not attended with rearrangement. 

Oxidation of A -cholestene-3-one in ether solution with 30% hydrogen peroxide and a catalytic amount of OSO4 gives both possible ei.s diols, which were isolated in the yields indicated. ... 

The Prevost reagent converts A -cholestene into both possible trans-diol dibenzoates. The diaxial 2/3,3a-isomer predominates over the diequatorial 2a,3/3-isomer in the ratio of 2 1, but difficulty in the separation limits the total yield to 31%. 

Rosenheim and Starling s oxidation of cholesterol (10) to A -cholestene-3/3,4/S-diol (11) involves attack a to the more highly substituted unsaturated carbon. The workup specified is to add 100 g. of sodium acetate and heat briefly to convert... 

Other examples of this novel method of cis-hydroxylation are reported by Cinsburg, Berkley et al., Klass et al. Slates and Wendler, Jefferies and Milligan, and Gunstone and Morris. In the c/s-hydroxylation of d -cholestene the yield of the 2 8-3)3-diol is improved from 50-65% to 81% by carrying out the reaction at 20° under nitrogen for 12 hrs. (standard procedure 45-90° for 1-20 hrs.)... 

During the 1960 s, the above sequence of reactions was confirmed by different in vitro studies. Mendelsohn and Staple showed that labelled cholesterol could be converted into 5j8-cholestane-3a,7a,12 -triol by 20000 X g supernatant fluid of rat liver homogenates [23]. The enzymatic conversion of cholesterol into 7a-hydroxy-cholesterol was first shown by Danielsson and Einarsson using the microsomal fraction fortified with NADPH [24]. The conversion of 7 -hydroxycholesterol into 7 -hydroxy-4-cholesten-3-one was found to be catalysed by the microsomal fraction fortified with NAD [25]. The latter steroid was converted into 7a,12a-dihydroxy-4-cholesten-3-one by the microsomal fraction and NADPH [26]. The conversion of 7 -hydroxy-4-cholesten-3-one and 7a,12a-dihydroxy-4-cholesten-3-one into the corresponding 3a-hydroxy-5/8-saturated steroids was catalysed by soluble NADPH-de-pendent enzymes [25,27,28]. Since Hutton and Boyd found that 4-cholestene-3 ,7 -diol was a product of 7a-hydroxy-4-cholesten-3-one in vitro [25], it was first... 

In most studies on 12 -hydroxylation, labelled 7a-hydroxy-4-cholesten-3-one has been used as the substrate [32], and it is believed to be the most important substrate also in vivo. It cannot be excluded that 5j8-cholestane-3a,7 -diol as well as 7a-hy-droxycholesterol are substrates for the 12a-hydroxylase in minor pathways in vivo. 

In a study by Ali and Elliott it was shown that 5a-cholestane-3 ,7a-diol was an even better substrate for the 12a-hydroxylase in rabbit liver microsomes than 7a-hydroxy-4-cholesten-3-one (156%) [104]. This reaction is probably of importance in the formation of allocholic add. The high specificity of the 12 -hydroxylase towards the coplanar 5a-sterol nucleus is also evident from the finding that allochenodeoxycholic acid can be converted into allocholic acid in rats, both in vivo and in vitro [105,106, Chapter 11]. Based on the known structural requirements of the 12a-hydroxylase, Shaw and Elliott prepared competitive inhibitors with different substitutions in the C,2 position [107]. The best inhibitor of those tested was found to be 5a-cholest-ll-ene-3a,7 ,26-triol. Theoretically, such inhibitors may be used to increase the endogenous formation of chenodeoxycholic acid in connection with dissolution of gallstones. 

The microsomal 26-hydroxylase in rat liver has a higher substrate specificity than the mitochondrial. Of a number of C27-steroids, only 5j8-cholestane-3a,7a,12a-triol, 5)8-cholestane-3a,7a-diol, 7a-hydroxy-4-cholesten-3-one and 7a,12a-dihydroxy-4-cholesten-3-one were 26-hydroxylated to a significant extent [126]. In addition to hydroxylation in the 26 position, 5)8-cholestane-3a,7a,12a-triol was hydroxylated by the microsomal fraction of rat liver in the 23, 24 , 24/8 and 25 positions [40]. The hydroxylation in the 25 position was about as efficient as that in the 26 position. 

The mitochondrial enzyme has a broad substrate specificity and catalyses 26-hydroxylation of a number of C27-steroids. The most important substrates in vivo are believed to be 5)8-cholestane-3a,7a-diol, 7a-hydroxy-4-cholesten-3-one and 5j8-cholestane-3a,7a,12a-triol. Bjorkhem and Gustafsson found that 5j8-cholestane-3a,7a,12a-triol and 7a-hydroxy-4-cholesten-3-one were the best substrates in rat liver mitochondria and that the least efficient 26-hydroxylation occurred with cholesterol as substrate [126,130]. There was also a small extent of 25-hydroxylation of cholesterol in the mitochondrial fraction [130]. The major part of the 26-hydroxylase is bound to the inner mitochondrial membranes [130,131]. Thus the hydroxylase activity is low with intact mitochondria and NADPH as cofactor. Under such conditions citric acid and isodtric acid, which are able to penetrate the inner mitochondrial membrane, stimulate 26-hydroxylation much more efficiently than NADPH [130,131]. It is evident that citric acid and isocitric acid generate NADPH inside the mitochondrial membrane. When using leaking mitochondria, NADPH stimulates the reaction about as efficiently as isocitrate [130,131]. 

Oftebro et al. reported that the mitochondrial fraction of a liver homogenate from a biopsy of a CTX patient was completely devoid of 26-hydroxylase activity [193]. The possibility that there had been a general inactivation of the mitochondrial fraction seems excluded since there was a significant 25-hydroxylase activity towards vitamin D,. There was a substantial accumulation of 5 -cholestane-3a,7a,12a-triol, the immediate substrate for the 26-hydroxylase in cholic acid biosynthesis. It was suggested that the accumulation of 5)3-cholestane-3a,7a,12a-triol would lead to increased exposure to the action of the microsomal 23-, 24- and 25-hydroxylases. The alternative 25-hydroxylase pathway would then be of importance for the formation of cholic acid in patients with CTX (Fig. 13). If the 25-hydroxylase pathway has an insufficient capacity, this would explain the accumulation of the different 25-hydroxylated intermediates in patients with CTX. A lack of the mitochondrial 26-hydroxylase would also lead to accumulation of intermediates in chenodeoxycholic acid biosynthesis such as 5)8-cholestane-3a,7a-diol and 7a-hy-droxy-4-cholesten-3-one. Such accumulation would lead to increased exposure to the microsomal 12a-hydroxylase which would yield a relatively higher biosynthesis of cholic acid. This would explain the marked reduction in the biosynthesis of chenodeoxycholic acid in patients with CTX. 

Results of various in vivo experiments with labelled bile acid precursors in patients with CTX have been published [185,190,195]. All these experiments show that there is a defect in the oxidation of the steroid side chain in the biosynthesis of cholic acid but are not fully conclusive with respect to the site of defect. Bjorkhem et al. administered a mixture of [ H]7a,26-dihydroxy-4-cholesten-3-one and [ " C]7a-hy-droxy-4-cholesten-3-one to a patient with CTX [195]. The ratio between and C in the cholic acid and the chenodeoxycholic acid isolated was 40 and 60 times higher, respectively, than normal. Similar results were obtained after simultaneous administration of H-labelled 5)3-cholestane-3a,7a,26-triol and 4- C-labelled 5j8-cholestane-3a,7a-diol. The results of these experiments are in consonance with the contention that the basic defect in CTX is the lack of the 26-hydroxylase, but do not per se completely exclude other defects in the oxidation of the side chain. 

L-Selectride was capable of equatorial attack on 3/ -acetyl-5-cholesten-7-one to give 5-cholesl-ene-3/ ,7a-diol 2. However, 7-oxo steroids have a particular tendency to undergo equatorial attack (see p 4034). 

Ailylic oxidation. The oxidation of cholesteryl benzoate with this complex to the A -7-ketone is remarkably fast (complete in less than 30 minutes) and the yield of ketone is routinely 70-75%. The oxidation of A -cholestene-3/J,5a-diol to the -7-ketone is complete in less than 2 minutes and again clean. However, oxidation of A -cholestene-3 8,5j3-diol is much slower and leads to many byproducts. The paper suggests two possible mechanisms for these oxidations, both of which involve prior attack at the double bond. 

Cholestanone, 497 5a-Cholestanone, 475 A -Cholestene, 497 A -Cholestene-3d,5a-diol, 110 A -Cholestene-3/3,5(3-diol, 110 Cholesteryl benzoate, 110 Chromenes, 235-236 A -Chromens, 407 Chromic acid-Silica gel, 110 Chromic anhydride-3,5-Dimethylpyrazole complex, 110 Chromium carbonyl, 110 Chromium(III) chloride-Lithium aluminum hydride, 110-112 Chromyl chloride, 112 ... 

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