Cholesterol 7a-hydroxylase

Cholesterol 7a-hydroxylase, a cytochrome P450-dependent enzyme, catalyzes the first and rate-limiting step in the biosynthesis of bile acids from cholesterol. Potential mechanisms for regulation of the enzyme have been extensively studied. 

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. 

Microsomes from rat liver were prepared in 100 mAf potassium phosphate buffer (pH 7.2) containing 100 mAf sucrose, 50 mAf KC1, 50 mAf NaF, 

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

These results are consistent with the conclusion that SCP2 provides a physiological means for providing cholesterol to cholesterol 7a-hydroxylase and thus modulating its activity by regulating the availability of substrate. 

We investigated the effect of homogeneous rat liver SCP2 on this process [33]. As shown in Fig. 4, the utilization of lipid droplet cholesterol for pregnenolone formation by adrenal mitochondria was dramatically increased by the addition of homogeneous SCP2. This effect was concentration dependent, and it was not mimicked by the addition of albumin. 

In order to determine the mechanism of this enhanced utilization of droplet cholesterol, the effect of SCPj on individual steps of the sterol transfer process and of mitochondrial utilization was tested. The ability of pure SCPj to bind unesterified cholesterol from lipid droplets was determined by incubation of lipid droplets with SCPj for 20 min at 37 C, followed by subsequent analysis of the levels of isotopic cholesterol remaining with the reisolated lipid droplets and that bound to SCPj in the droplet-free subnatant fraction. There was a linear increase in the amount of subnatant radioactivity with increasing amounts of SCPj added to the lipid droplets 

From the data shown (Fig. 5C and D), the molar ratio of cholesterol depletion from the lipid droplets or cholesterol accumulating in mitochondria to the quantity of SCP2 added was 1.01 0.05. Thus, the stoichiometry of transport of cholesterol from lipid droplets to AG-blocked mitochondria, conducted by SCP2, is 1/1. 

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Matheson, H. B., Colon, I. S., and Story, J. A. (1995). Cholesterol 7a-hydroxylase activity is increased by dietary modification with psyllium hydrocolloid, pectin, cholesterol and cholestyramine in rats. /. Nutr. 125,454-M58. 

Deficiency of cholesterol 7a-hydroxylase can increase LDL in the heterozygous state. Homozygotes can also have elevated triglycerides, resistance to reductase inhibitors, and increased risk of gallstones and coronary disease. Autosomal... 

The initial steps in BA synthesis are characterised by the introduction of a hy-droxylic group in the la position, or in position 27, followed by another in the la position into the cholesterol nucleus. Both synthetic pathways (the neutral and the acidic pathways) possess a distinct microsomal 7-oxysterol hydroxylase, which is regulated by different genes. The most recently described disorder of BA synthesis is cholesterol 7a-hydroxylase deficiency, in which their decreased production through the classical pathway is partially balanced by activation of the alternative pathway. Cholesterol levels increase in the liver, with a consequent low-density lipoprotein hypercholesterolemia, and cholesterol gallstones may result, although there is no liver disease. In contrast, a defect in the conversion of 27-hydroxy-cholesterol to la,27-dihydroxy-cholesterol due to deficiency of the oxysterol 7a-hydroxylase specific for the alternate pathway, causes severe neonatal liver disease [8]. 

Rudel, L.L., Deckelman, C., Wilson, M.D., Scobey, M., and Anderson, R. 1994. Dietary cholesterol and downregulation of cholesterol 7a-hydroxylase and cholesterol absorption in African green monkeys. J. Clin. Invest. 93, 2463-2472. 

ATP synthesis protein 8 HMG CoA-reductase Cholesterol 7a-hydroxylase ATP synthesis X chain... 

The properties of 7a-hydroxylase from pigeon liver microsomes305 and from rat liver306,307 have been further described, and new assay methods are available.308,309 Free cholesterol, rather than a cholesteryl ester, was the preferred substrate for the enzyme from rat liver microsomes,310 and the substrate pool for the hydroxylase was about one third of the total amount of cholesterol present in the microsomal preparation.309 Cholesterol 7a-hydroxylase activity is more sensitive to thyroid function than are the activities of the enzymes responsible for cholesterol synthesis,311 and (22f )-22-aminocholesterol, although having no effect on serum or liver cholesterol levels in rats, drastically reduced 7a-hydroxylase activity.312... 

Patients with familial hypercholesterolaemia exhibit lower levels of plasma cholesterol after an operation for portacaval anastomosis, and it has now been shown in rats that such an operation causes an increase in HMG-CoA reductase and cholesterol 7a -hydroxylase activities. Many transplantable human and rodent hepatomas do not control the rate of sterol biosynthesis and HMG-CoA reductase levels in response to dietary cholesterol as normal liver cells do. However, certain hepatoma cells have now been found that, although lacking feedback regulation of choles-terologenesis in vivo, retain their regulatory ability in vitro It thus appears that malignant transformation is not necessarily linked to the loss of regulation by the cell of HMG-CoA reductase activity or sterol synthesis. 

For obvious reasons, inhibitors of cholesterol biosynthesis have aroused intense interest. Studies on a variety of species (mostly rats) have been made using 1-alkylimidazoles," colchicine," and chenodeoxycholic acid, the last work being particularly interesting as the metabolite affected HMG-CoA reductase but not cholesterol 7a-hydroxylase, the steps believed to be rate-limiting for the biosynthesis of sterols and of bile acids respectively. Arsenite, /8-mercaptoethanol, dithiothreitol, and ethanethiol all inhibited the biosynthesis of cholesterol from MVA in rat liver homogenates. The accumulation of 4,4-dimethyl-5a-cholest-8-en-3/3-ol together with the corresponding A -diene supported the view that... 

Improved methodology for the rapid assay of hepatic HMG-CoA reductase has been described and new and simplified assays are available for cholesterol 7a-hydroxylase, 4-methylsterol oxidase, 3/3-hydroxy-steroid dehydrogen-ase, the biosynthesis of bile acids, and microsomal cholesterol levels.The rate of biosynthesis of gibberellins has been monitored by a bioassay based on /3-amyrin production of Amaranthus seeds. 

Axelson M, Bjorkhem I, Relhner E, Einarsson K. The plasma level of 7a-hydroxy-4-cholesten-3-one reflects the activity of hepatic cholesterol 7a-hydroxylase in man. Febs Lett 1991 284 216-8. 

Cholesterol 7a-hydroxylase (CYP7A1) The mixed-function oxidase cytochrome P450 enzyme catalyzing the initial, rate-limiting step for conversion of cholesterol to bile acids. 

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