7a-hydroxycholesterol ,

In 3/i-hydroxysteroid-A5-oxidoreductase deficiency, 7a-hydroxycholesterol undergoes side-chain oxidation, producing 3/l,7a-dihydroxy-5-cholenoic acid and... 

In work published in 1989, Hylemon et al. used cholesterol oxidase to convert 7a-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one. Cholesterol that remained was converted to 4-cholesten-3-one. 7/3-Cholesterol, which was added as an internal steroid recovery standard, was oxidized to 7/3-hydroxy-4-cholesten-3-one. These steroid products were analyzed by Qg reversed-phase chromatography on an Altex Ultrasil-ODS column (4.6 mm x 25 cm) using 70 30 (v/v) mixture of acetonitrile and methanol (Fig. 9.83). The eluate was monitored at 240 nm, and the amount of product determined from a calibration curve. 

The second step in the synthesis of bile acids, according to Hylemon et al. (1991), is the conversion of 7a-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one by NAD+-dependent 3/3-hydroxy-A5-C27-steroid oxidoreductase. This enzyme is located in the endoplasmic reticulum of liver, and its catalysis of the 3/3-hydroxy group also results in isomerization of the double bond from A5 to A4. 

The probable mechanism of the synergetic effect of L-ascorbic acid and substances capable of binding bile acids in the gastrointestinal tract is shown in Scheme 1. An increased intake of L-ascorbic acid accelerates the formation of 7a-hydroxycholesterol and thereby also accelerates the overall rate of cholesterol transformation into bile acids. Bile acids excreted from the liver into the gastrointestinal tract become bound and thus are prevented from affecting 7 -hydroxylation of cholesterol through a feedback mechanism. In this way, a permanent disequilibrium occurs between cholesterol and bile acids with increased cholesterol transformation to bile acids and enhanced irreversible output of the products of this reaction, which results in a decline of cholesterol levels in the blood and tissues. 

Substitution, Elimination, and Solvolysis.—7a-Hydroxycholesterol, an intermediate in the enzymic formation of bile acids from cholesterol, has been difficult to obtain by chemical methods. It is now available from the accessible 7/3-isomer by reaction with HBr at —78°C, which affords 7a-bromocholesteryl benzoate (21), followed by acetolysis (KOAc-HOAc) to form the 7a-acetoxy-derivative (22). 

The initial and rate-limiting step of bile acid synthesis is oxidation of cholesterol to 7a-hydroxycholesterol by a mixed function oxidase from the cytochrome P450 superfamily, cholesterol 7a-hydroxylase (CYP7A1). 

Cholesterol 7a-hydroxyIase has been partially purified from rat and rabbit liver (H5). The enzyme is located in the smooth endoplasmic reticulum and is dependent on cytochrome F-450 and NADPH-cytochrome P-450 reductase for activity (H5). The particular cytochrome P-450 associated with microsomal cholesterol 7a-hydroxylase activity constitutes a small fraction of total liver cytochrome P-450 and, in the rabbit, it appears to be a subfraction of cytochrome P-450lm4 (B28). Measurement of the activity of this enzyme by isotope incorporation is complicated by dilution of added cholesterol by endogenous microsomal cholesterol. A method has now been developed to remove cholesterol fit>m microsomes, so that the mass of 7a-hydroxycholesterol formed during enzyme assay can be accurately calculated (S25). Using this assay, cholic acid feeding was shown to suppress the activity of cholesterol 7a-hydroxylase in rat liver, whereas cholesterol feeding did not (S25). 

Other clinical signs consist of progressive neurologic dysfunction, cataracts, and premature atherosclerosis (SI). The disease is inherited as an autosomal recessive trait, but is usually only detected in adults when cholesterol and cholestanol have accumulated over many years (S2). Biochemical features of the disease include striking elevations in tissue levels of cholesterol and cholestanol and the presence of unusual bile acids, termed bile alcohols, in bile. These bile alcohols are mainly 5 -cholestane-3a,7a,12a,24S, 25-pentol, Sp-diolestane-3a,7a,12a,23 ,25-pentol and 5P-du)lestane-3a,7a,12a,25-tetrol (S2). As chenodeoxycholic acid is deficient in the bile of patients with CTX, it was postulated that early bile salt precursors are diverted into the cholic acid pathway and 12a-hydroxy bile alcohols with an intact side chain accumulate because of impaired cleavage of the cholesterol side chain and decreased bile acid production (S2). HMG-CoA reductase and cholesterol 7a-hydroxylase activity are elevated in subjects with CTX (N4, N5), so that sufficient 7a-hydroxycholesterol should be available for bile acid synthesis. 

G8. Goldman, M., Vlahcevic, Z. R., Schwartz, C. C., Custafsson, J., and Swell, L., Bile acid metabolism in cirrhosis. VIII. Quantitative evaluation of bile acid synthesis from [TR- H] 7a-hydroxycholesterol and [G- H]26-hydroxycholesterol. Hepatology 2, 59-66 (1982). 

Cholesterol and many of its biosynthetic precursors are highly insoluble in aqueous media. Yet, cholesterol biosynthesis, utilization and intracellular transfers occur in environments which involve both aqueous and nonaqueous components. For example, the enzymes involved in the conversion of squalene to cholesterol, the conversion of cholesterol to cholesterol esters, and the conversion of cholesterol to 7a-hydroxycholesterol are associated with the endoplasmic reticulum (microsomes). The conversion of cholesterol to pregnenolone, an essential first step in steroid hormone biosynthesis, occurs in mitochondria. In addition, transfers of cholesterol from cytoplasmic lipid inclusion droplets through the cytosol to the mitochondria are essential for steroid hormone production. 

The conversion of cholesterol to 7a-hydroxycholesterol is the first step and the major regulatory step in bile acid formation (Chapter 9). 

Fig. 8 is a schematic diagram of a cell which shows the known sites in which sterol carrier proteins are involved in cholesterol biosynthesis, utilization and intracellular transfer. SCP, participates in the conversion of squalene to lanosterol and SCP2 participates in the conversion of lanosterol to cholesterol, the conversion of cholesterol to cholesterol ester by ACAT, and probably also in the conversion of cholesterol to 7a-hydroxycholesterol. SCPj transfers cholesterol from cytoplasmic lipid inclusion droplets to mitochondria in the adrenal and SCPj also translocates cholesterol from the outer to the inner mitochondrial membrane. 

A coupling appears to exist between the rate-limiting enzyme in the biosynthesis of cholesterol, HMG-CoA reductase, and cholesterol 7a-hydroxylase. The two enzymes seem to be located close to each other on the endoplasmic reticulum membrane [66], and the two activities covariate under most conditions. Results from both in vivo and in vitro experiments show that newly synthesized cholesterol is the preferred substrate for cholesterol 7a-hydroxylase. In an early study by Staple and Gurin, it was shown that the bile acids in bile had a higher radioactivity than cholesterol after administration of labelled acetate to rats [67]. Bjorkhem and Danielsson found that the specific radioactivity of 7a-hydroxycholesterol was higher than that of cholesterol after incubation of labelled mevalonate with the 10000 x g supernatant fluid of a rat liver homogenate [50]. Balasubramaniam et al. showed that 7a-hydroxycholesterol isolated from the livers of rats after intravenous administration of labelled cholesterol had a lower specific radioactivity than cholesterol [58]. Cronholm and collaborators measured the incorporation of isotope from [1- H2]-,... 

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... 

Yamasaki et al. have studied a pathway for formation of chenodeoxycholic acid in the rat involving intermediate formation of 7a-hydroxycholesterol, 3 -hydroxy-5-cholenoic acid and 7a-hydroxy-3-oxo-4-cholenoic acid [176-179]. In this pathway, changes in the side chain occur after the rate-limiting step. From the data available, it is not possible to evaluate the quantitative importance of this pathway. 

The possibility that the biosynthesis of bile acids is regulated by a negative feedback mechanism was supported by early experiments by Thompson and Vars [206] and Eriksson [207], who showed that the rate of bile acid synthesis in rats increased about 10-fold when a bile fistula is made. Bergstrom and Danielsson demonstrated that duodenal infusion of taurochenodeoxycholic acid in bile fistula rats restored the increased synthesis to a normal rate [208]. Danielsson et al. [44] showed that the cholesterol 7a-hydroxylase activity increased in parallel with the bile acid synthesis after cannulation of the bile duct in rats. In a subsequent work by Mosbach et al., it was reported that the incorporation of isotope from labelled acetate, mevalonate and cholesterol but not from labelled 7a-hydroxycholesterol into bile acids was inhibited by duodenal infusion of taurocholate to bile fistula rats [209]. The incorporation of isotope from labelled acetate, mevalonate and cholesterol but not from labelled 7a-hydroxycholesterol was stimulated in perfused livers of cholestyramine-treated rabbits [210]. It was concluded that there are essentially no rate-limiting steps beyond 7a-hydroxycholesterol in the biosynthesis of bile acids from acetate. Since both cholesterol and bile acid biosynthesis was subjected to negative feedback inhibition by bile acids, it cannot be excluded that inhibition of cholesterol biosynthesis precedes inhibition of the bile acid biosynthesis, and that the latter inhibition is secondary to the former. 

Mitropoulos et al. have measured the rate of excretion and the specific activities of cholic acid and chenodeoxycholic acid in bile fistula rats fed [ H]cholesterol and infused with [ " C]mevalonate or [ C]7a-hydroxycholesterol [255]. It was concluded that newly synthesized hepatic cholesterol was the preferred substrate for the formation of cholic acid. It could not be excluded, however, that part of the chenodeoxycholic acid had been formed from a pool of cholesterol different from that utilized in cholic acid biosynthesis. The mitochondrial pathway, starting with a 26-hydroxylation, could have accounted for a significant fraction of the chenodeo-... 

Dihydroxy-5-cholenic acid, postulated as an intermediate in the mitochondrial synthesis of chenodeoxycholate from 7a-hydroxycholesterol [180,181], and a closely allied product, 3a,7a-dihydroxy-4-cholenic acid, have each been isolated from bile of hens and humans. Incubation of 3)8,7a-dihydroxy-5-cholenic acid with rat or carp hepatic microsomal preparations fortified with NADPH provided 3-oxo-7a-hydroxy-4-cholenic acid. Administration of the A acid to rats with cannulated bile ducts provided biliary metabolites identified as chenodeoxycholate, a- and )8-muricholates. Similar incubation of 3-oxo-7a-hydroxy-4-cholenic... 

Another hydroxylation role for vitamin C in the hepatic microsomal fraction is the stepwise conversion of cholesterol to the bile acid, cholic acid, via 7a-hydroxycholesterol, 3a,7a-dihydroxycoprostane and 3a,7a,12o-trihydroxycoprostane. Also, in lipid metabolism, conventional fatty acids with an even number of carbon atoms are a-oxidised by a mono-oxygenase and subsequently decarboxylated to form an odd-numbered carbon derivative and both these steps appear to require ascorbic acid. As the initial a-oxidation is brought about by a... 

The most abundant oxysterols in human plasma are 27-, 24-, and 7a-hydroxycholesterol (Fig. 5), and most of these are esterified to fatty acids at the 3p position by lecithinxho-lesterol acyltransferase [19]. Both unesterified and esterified oxysterols partition in... 

Figure 2.13a Fragmentation of GP-tagged oxysterols illustrated by 24S,25-epoxycholesterol. (a) Major fragments observed in MS spectra recorded on an ion trap, (b) Major fragment observed in MS spectra recorded on an ion trap. MS/MS spectra recorded on tandem quadrupole or Q-TOF instruments are a composite of (a) and (b). (c) The effect of 7a-hydroxylation on fragmentation as illustrated by 7a-hydroxycholesterol. (Continued)...

Steroid standards were purchased from the following sources Cholestane - 3/9,5a, 6/9-triol and 7a-hydroxycholesterol from Research Plus Laboratories, Inc. (Denville, NJ) Cholesterol, 25-hydroxycholesterol, 7-... 

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