Cholesterol triol

As mentioned earlier, oxidation of LDL is initiated by free radical attack at the diallylic positions of unsaturated fatty acids. For example, copper- or endothelial cell-initiated LDL oxidation resulted in a large formation of monohydroxy derivatives of linoleic and arachi-donic acids at the early stage of the reaction [175], During the reaction, the amount of these products is diminished, and monohydroxy derivatives of oleic acid appeared. Thus, monohydroxy derivatives of unsaturated acids are the major products of the oxidation of human LDL. Breuer et al. [176] measured cholesterol oxidation products (oxysterols) formed during copper- or soybean lipoxygenase-initiated LDL oxidation. They identified chlolcst-5-cnc-3(3, 4a-diol, cholest-5-ene-3(3, 4(3-diol, and cholestane-3 3, 5a, 6a-triol, which are present in human atherosclerotic plaques. 

Fig. 5.3.6 Diagnose of Smith-Lemli-Opitz syndromesyndrome (SLOS). Urinary dehydropreg-nanetriols (DHPT 5/ -pregn-7-ene-3a,17a,20a-triol and 5j3-pregn-8-ene-3a,17a,20a-triol) partially replace PT in 7-dehydrocholesterol reductase deficiency and can be used to diagnose SLOS. The ratio of DHPT/PT correlates with dehydrocholesterol (DHC)/cholesterol (C) in affected patients (n = 34) and also correlates with clinical severity...

Figure 18.3. Scheme for the formation of cholesterol epoxides. Compounds are as follows (1) CHOL (2) a-epoxide (3) ft-epoxide (4) triol. For abbreviations, see Table 18.1. 

Cholesterol and ester derivatives Corticosteroids (ouabain, strophantidin, 4-pregnene-6b, 1 1 b,21-triol-3,20-dione)... 

C,2iS- H,3R]mevalonate, Bloxham and Akhtar showed that a tritium atom was lost whereas when [3a- H,26,27- C2]lanosterol was used the tritium was retained. The latter result was also observed by Hornby and Boyd. Presumably NAD is necessary for the oxidation at C-3 to a ketone prior to decarboxylation. Similarly, Miller and Gaylor showed that 4a-methyl-5a-cholest-7-en-3) -ol was oxidized only as far as the 4a-carboxylic acid, with retention of tritium at C-3 but loss from a 4a-C H3 group. In the latter case, the recovered 4a-methyl sterol showed no sign of tritium enrichment due to isotope effects. In banana, alkylation at C-24 seems to precede loss of the 4a-methyl groups. When the rat liver system was inhibited by cholestane-3, 5a,6 -triol, sterols accumulated which retained a methyl group at C-4. Both 4,4-dimethyl- and 4 -methyl-cholest-8-en-3/I-ol Uere isolated, and were shown to be converted into cholesterol under normal conditions. 

In summary, these studies demonstrated that in CTX the impaired synthesis of bile acids is due to a defect in the biosynthetic pathway involving the oxidation of the cholesterol side-chain. As a consequence of the inefficient side-chain oxidation, increased 23, 24 and 25-hydroxylation of bile acid precursors occurs with the consequent marked increase in bile alcohol glucuronides secretions in bile, urine, plasma and feces (free bile alcohols). These compounds were isolated, synthesized and fully characterized by various spectroscopic methods. In addition, their absolute stereochemistiy determined by Lanthanide-Induced Circular Dichroism (CD) and Sharpless Asymmetric Dihydroxylation studies. Further studies demonstrated that (CTX) patients transform cholesterol into bile acids predominantly via the 25-hydroxylation pathway. This pathway involves the 25-hydroxylation of 5P-cholestane-3a,7a, 12a-triol to give 5P-cholestane-5P-cholestane-3a,7a,12a,25- tetrol followed by stereospecific 24S-hydroxylation to yield 5P-cholestane-3a,7a,12a,24S,25-pentol which in turn was converted to cholic acid. 

A solution of 1 g. of cholesterol a-oxide in 30 ml. of hot acetone is treated with a solution of 0.625 g. of periodic acid dihydrate in 10 ml. of water." Before all the precipitated oxide has redissolved, thin plates of cholestane-3/3, 5a,6(8-triol begin to separate. The mixture is refluxed for one half hour, cooled, and the product collected yield 0.83 g. (81%), m.p. 231-232°. A second crop of material (0.14 g.) melted at 225-226°. 

From the above investigations, summarized in Fig. 2, it was concluded that 7a-hydroxylation of cholesterol may be the first step in the conversion of cholesterol into bile acids, and that 5/S-cholestane-3a,7a,12a-triol probably is an intermediate in cholic acid formation. Since 5)S-cholestane-3a,7a-diol was rapidly converted into chenodeoxycholic acid and only to a small part into cholic acid [19], it was concluded that 5i8-cholestane-3a,7a-diol is a corresponding intermediate in the formation of chenodeoxychohc acid. Samuelsson showed that the conversion of cholesterol into bile acids most probably involves a ketonic intermediate, since [3a- H]cholesterol lost its tritium when converted into chohc acid [1,20]. Since... 

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... 

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]. 

Gustafsson has reported that Ca " and Mg " have different effects on mitochondrial 26-hydroxylation of endogenous cholesterol, exogenous cholesterol and 5j8-cholestane-3o,7a,12a-triol [132]. He concluded that there might be different transport mechanisms for the two substrates through the mitochondrial membranes. This was later supported by studies by Pedersen et al., showing that the stimulatory effect of Mg was similar for hydroxylation of several substrates catalysed by a partially purified system [133]. 

The mitochondrial 26-hydroxylase is inhibited by biliary drainage and is not influenced by starvation or treatment with phenobarbital [126]. Gustafsson reported that the mitochondrial 26-hydroxylation of cholesterol, 7a-hydroxycholesterol and 7a-hydroxy-4-cholesten-3-one was stimulated whereas 26-hydroxylation of 5)8-cholestane-3a,7a,12a-triol was inhibited by biliary obstruction [127]. Whether the... 

Conclusive evidence that a species of cytochrome P-450 was involved in the hydroxylation was presented by Okuda et al., who showed that the photochemical action spectrum for reversal of the carbon monoxide inhibition of 26-hydroxylation of 5)8-cholestane-3a,7a,12a-triol in rat liver exhibited a maximum at 450 nm [134]. Pedersen et al. [135] and Sato et al. [136] reported simultaneously that small amounts of cytochrome P-450 could be solubilized from the inner membranes of rat liver mitochondria that was active towards cholesterol as well as 5)8-cholestane-3a,7a,12a-triol in the presence of ferredoxin, ferredoxin reductase and NADPH. The mechanism of hydroxylation is thus the same as that operative in the biosynthesis of steroid hormones in the adrenals and in the la-hydroxylation of 25-hydroxyvitamin D in the kidney (Fig. 8). The liver mitochondrial cytochrome P-450 was not active in the presence of microsomal NADPH-cytochrome P-450 reductase [135,136]. Ferredoxin reductase as well as ferredoxin were active regardless of whether they were isolated from rat liver mitochondria or bovine adrenal mitochondria [133]. The partially purified cytochrome P-450 had a carbon monoxide difference spectrum similar to that of microsomal cytochrome P-450 from liver microsomes and adrenal mitochondria. In the work by Pedersen et al. [133], the concentration of mitochondrial cytochrome P-450 in rat liver mitochondria from untreated rats was calculated to be only about 0.1 nmole/mg protein. Treatment of rats with phenobarbital increased the specific content of cytochrome P-450 in the mitochondria more than 2-fold, without significant increase in the 26-hydroxylase activity. The carbon monoxide spectrum of the reduced cytochrome P-450 solubilized from liver mitochondria of phenobarbital-treated rats exhibited a spectral shift of about 2 nm as compared to the corresponding spectrum obtained in analysis of preparations from untreated rats. This was taken as evidence that more than one species of cytochrome P-450 was present in the preparation. It was later shown by Pedersen et al. [137] and Bjbrkhem et al. [138] that the preparation was also able to catalyse 25-hydroxylation of vitamin D3 and that different enzymes are involved in... 

The major bile salt of the carp, Cyprinm carpio, is 5a-cyprinol sulfate [21]. When [4- C]cholesterol was injected intraperitoneally into the carp, radioactive 5a-cyprinol was isolated from gallbladder bile [148]. It has been shown that the initial step in the major pathway for the formation of 5a-cyprinol (VI) from cholesterol (XV) is the 7a-hydroxylation of cholesterol to form cholest-5-ene-3j8,7a-diol (XVI) [149] (Fig. 4). It has also been shown that the double bond is isomerized to the A position before being reduced [150]. These in vivo studies suggest that until the intermediary formation of a A compound, presumably 7 ,12a-dihydroxycholest-4-en-3-one (XVII), the sequence of reactions in the biosynthesis of 5 -cyprinol (VI) in the carp is the same as that in the conversion of cholesterol (XV) to cholic acid (XIV) in mammals. 7a,12a-Dihydroxycholest-4-en-3-one (XVII) was found to be converted into 5a-cholestane-3a,7a,12a-triol (XVIII) by the microsomal fraction of carp hver fortified with NADPH [151]. The conversion of the triol (XVIII) to 5a-cyprinol (VI) via 27-deoxy-5a-cyprinol (XIX) was also established. The 26-hydroxylation of the triol (XVIII) was catalyzed by the microsomal fraction fortified with NADPH, and the 27-hydroxylation of 27-deoxy-5a-cyprinol (XIX) was catalyzed by the mitochondrial fraction fortified with NADPH [151]. 

Hoshita et al. have shown that liver microsomes from the green iguana, in which the major biliary bile salt is tauroallocholate, convert 7a,12a-dihydroxycholest-4-en-3-one (XVII) into 5a-cholestane-3a,7a,12a-triol (XVIII) rather than into 5)8-choles-tane-3 ,7a,12a-triol (VIII) which is involved in cholic acid biosynthesis [164]. On the basis of this result and that obtained from studies with carp liver [151], it can be assumed that 5a-bile acids and alcohols are formed from cholesterol by a modification of the biosynthetic pathway to the corresponding 5y8 isomers in which the only difference is the stereospedfic saturation of the A double bond of the intermediate XVII. 

Selective removal of bromine from 3/3,5,6/3-tribromo-5a-cholestane to give 3/3-bromocholest-5-ene was achieved by reaction with [T -C5H5Cr(N02)2]2. The reactions of 11/3-hydroxy-steroids with dialkylaminosulphur trifluorides depend on the substitution at C-9 and involve the formation of intermediate (11) (Scheme 1) (see ref. 232). Selective dehydration with FeCls adsorbed on silica gel allowed the conversion of 5a-cholestane-3/3,5-diol into cholesterol (80%) and 3i3-acetoxy-5a-cholestane-5,25-diol into 3/S-acetoxycholest-5-en-25-ol (72%). Other examples and additionally the hydrolysis of 5,6a-epoxy-5o -cholestan-3/3-ol to the 3/3,5a,6/8-triol (90%) were reported. Chromatographic alumina is reported to effect smooth elimination of sulphonic acids from the esters with less than normal rearrangement. Thus lanosteryl tosylate and cycloartenyl tosylate gave the respective A -compounds in yields of 90% and 45% respec-... 

In 1963, cholesterol hydroperoxides were reported (36) in egg-containing foods irradiated by sunlight. Chicoye et al. (37) observed the following 5 photoxidation derivatives of cholesterol in spray-dried yolk exposed to either 40-watt fluorescent lamp (approx. 280 hours) or summer sunlight (5 hours) 33"hydroxy-cholest-5-en-7-one (7-keto) cholest-5-ene-33,7a-diol (7a-diol) cholest-5-ene-33,73-diol (73-diol) 5,63-epoxy-5a-cholestan-33-ol (3-epoxide) and 5a-cholestane-33, 5cx, 63-triol (triol). Subsequently, Tsai et al. (38) developed methodology to demonstrate the presence of 5,6a-epoxy-5a-cholestan-33-ol (a-epoxide) in dried egg products which were spiked with the epoxides. 

In a sequel, Peng et al. (41) purified known oxidation products of cholesterol and studied their toxicity in cultured rabbit aortic smooth muscle cells. Using thin layer chromatography for separation followed by UV detection in 50% aqueous sulfuric acid spray, the oxidation products separated into 6 fractions according to their mobilities and were used in the cell culture. Potent cytoxic effects were noted for a number of the identified cholesterol oxidation products. In contrast, purified cholesterol at the same concentration produced no toxic effects. The results demonstrated that 25-hydroxycholesterol and triol were the most toxic agents. Peng et al. (42) confirmed and... 

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