Biosynthesis of cholestanol in higher plants

To understand the early steps of C(27) brassinosteroid biosynthesis, metabolic experiments were performed with Arabidopsis thaliana and Nicotiana tabacum seedlings, and with cultured Catharanthus roseus cells. [26, 28-2H(6)]Campestanol, [26-2H(3)]cholesterol, and [26-2H(3)]cholestanol were administered to each plant, and the resulting metabolites were analyzed by gas chromatography-mass spectrometry. In all the species examined, [2H(3)]cholestanol was identified as a metabolite of [2H(6)]campestanol, and [2H(3)]cholest-4-en-3-one and [2H(3)]cholestanol were identified as metabolites of [2H(3)]cholesterol. This study revealed that cholestanol (C(27) sterol) was biosynthesized from both cholesterol (C(27) sterol) and campestanol (C(28) sterol). It was also demonstrated that cholestanol was converted to 6-oxocholestanol, and campestanol was converted to 6-oxocampestanol.     

Chromatographic separation of putative precursors of cholestanol

This paper describes convenient syntheses for labeled and unlabeled cholest-5-en-3-one, cholest-4-en-3-one, epicholesterol, cholest-4-en-3 beta-ol, and cholest-4-en-3 alpha-ol. The thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography of these compounds and of cholestanol and epicholestanol are also described. The synthesized compounds are potential precursors in the biosynthesis of cholestanol in mammals.     

Comparison of the metabolism of cholesterol, cholestanol, and -sitosterol in L-cell mouse fibroblasts

The following data have been obtained from comparative studies on the metabolism of cholesterol, cholestanol, and beta-sitosterol by L-cell mouse fibroblasts. (1) When the sterols are added to the growth medium under similar conditions, cellular incorporation of cholesterol > cholestanol > beta-sitosterol; (2) only limited cellular esterification of these compounds occurs; (3) no metabolic products arising from the sterols could be detected; (4) influx of all sterols is dependent upon the concentration; and (5) exogenous cholesterol reduces mevalonate incorporation into cellular sterol to a lesser extent than acetate or glucose. The metabolism of these sterols is discussed in relation to their ability to influence de novo sterol biosynthesis.     

Esterification of cholesterol and cholestanol in the whole body,tissues, and frass of Heliothis zea

The quantity of free and esterified sterols in the whole body, intestine, hemolymph, fat body, and frass of 6th-instar larvae of H. zea, fed cholesterol or cholestanol, was measured in order to determine if there was a difference in the utilization of these two molecules. The principal sterol in the tissues of the larvae was cholestanol or cholesterol, when they were fed diet containing these two molecules, respectively; there was little, if any, metabolism of dietary cholestanol to cholesterol. There was little or no difference in the amount of total sterol in the whole body, tissues, or frass of larvae fed the two different diets, indicating that the absence of a Δ⁵-bond in cholestanol does not prevent the uptake or distribution of this sterol to various tissues. However, the relative percentage of steryl ester was significantly higher in prepupae reared on a diet containing cholestanol instead of cholesterol (6–7-, 4-, 13-, 4-, and 2-fold increase, for the whole body, intestine, hemolymph, fat body, and frass, respectively). The average percentage of total sterol that was esterified in the tissues was greater in the fat body (10.8 ± 15.4 and 44.2 ± 12.3%, respectively, for larvae fed cholesterol and cholestanol) than in the hemolymph (0.5 ± 0.1 and 6.3 ± 0.8%) and intestine (1.2 ± 0.1 and 4.7 ± 1.1%). The percentage of sterol that was esterified in the frass of larvae was large (26.9 ± 3.7 and 48.2 ± 0.5%, respectively, for larvae fed cholesterol and cholestanol). Therefore, the fact that larvae of H. zea fed cholestanol, instead of cholesterol, contain this saturated molecule as their principal tissue sterol and preferentially esterify it may explain, at least in part, why their rate of growth on cholestanol is slower than on cholesterol.     

Effect of cholestanol feeding on sterol concentrations in the serum, liver, and cerebellum of mice

In order to elucidate the mechanism of xanthoma formation in cerebrotendinous xanthomatosis, mice were fed for 32 weeks with a diet rich in 5 alpha-cholestan-3 beta-ol (cholestanol) (1%, w/w). The concentrations of sterols in the serum, liver, and cerebellum were determined using high performance liquid chromatography. In the cholestanol-fed mice, the cholestanol concentrations in the serum and liver reached maxima in the first 2 to 4 weeks; the levels were about 30- to 100-fold higher than in the control diet mice. The cholestanol concentrations declined thereafter, finally to 50-60% of the maxima. Cholesterol concentrations were slightly lower in the cholestanol-fed mice throughout the experiments than in the control diet mice. On the other hand, the levels of cholestanol in the cerebellum increased almost linearly in parallel to the feeding time, and no decline was observed. These results suggest that the capacity of the liver to remove or degrade cholestanol was increased by long-term intake of this compound, whereas the cerebellum had no such feed-back regulation. Histological examinations using an electron microscope revealed the enlargement of lysosomal granules in the liver of the cholestanol-fed mice.     

Effects of cholestanol feeding on corneal dystrophy in mice

A cholestanol-enriched diet administered for 8 months to BALB/c mice produced in 20% two kinds of corneal opacities resembling calcific band keratopathy and Schnyder's crystalline dystrophy in humans. The concentrations of cholestanol in serum, liver and cornea of the corneal opacity bearing mice were 30-40-times higher than those of normal mice. On the other hand, brain cholestanol level increased only 7-times in the opacity group as compared with that of control group. There was no significant difference in the cholesterol concentrations of serum and several tissues among opacity, non-opacity and the control group. The crystal particles were observed between epithelial basement membrane and superficial stroma by the electron microscopy. Energy dispersive analysis of the particles revealed that the deposits were composed principally of calcium and phosphorus with other crystalline materials, which was presumed to be cholestanol. These results suggest that the cholestanol may deposit in the cornea from elevated serum levels. Deposition of cholestanol in cornea and related area may be a cause of corneal dystrophy in CTX.     

Uptake of cholesterol and cholestanol by the intestine,hemolymph, and fat body of Heliothis zea

The effect of the concentration and structure of dietary sterol on its uptake and distribution in the intestine, hemolymph and fat body was studied in sixth-instar larvae of Heliothis zea. When cholesterol (cholest-5-en-3β-ol) was inoculated per os into the foregut of larvae, it was rapidly taken up by the intestine. Some of the dietary sterol then passed into the hemolymph, primarily via the midgut, during at least the first 9 h after inoculation, while at least 7% of the dose remained associated with the intestine. The amount of dietary sterol per 0.10 g of hemolymph increased until it reached 3–6% of the dose after 9 h. The amount of sterol per 0.10 g of the fat body increased to as much as 5% of the dose after 10 h. As the concentration of sterol in the dose increased from 0.3 to 15 μg/4 μl, the amount of sterol associated with the intestine, hemolymph, and fat body also increased. When cholesterol was inoculated intrahemocoelically, instead of per os, the amount of sterol in the hemolymph decreased, for at least the first 8 h after inoculation, and may have been absorbed, at least in part, by the intestine. The absence of a double bond in cholestanol (5α-cholestan-3β-ol) had no significant effect, at least 5 h after inoculation, on the uptake and distribution of this sterol in the intestine, hemolymph, and fat body of the larva. The results of this study indicate that although larvae of H. zea fed cholestanol have a slower rate of growth than those fed cholesterol, this may be due to differences in the utilization of the two sterols rather than to differences in their uptake by the tissues.     

Biosynthesis of cholestanol: 5-alpha-cholestan-3-one reductase of rat liver

The 3-beta-hydroxysteroid dehydrogenase of rat liver which catalyzes the conversion of 5alpha-cholestan-3-one to 5alpha-cholestan-3beta-ol is localized mainly in the microsomal fraction. The enzyme required NADPH as hydrogen donor and differed from the known 3-beta-hydroxysteroid dehydrogenases of the C(19) series in being inactive in the presence of NADH. The microsomal preparations did not reduce the 3-keto groups of cholest-4-en-3-one, cholest-5-en-3-one, or 5beta-cholestan-3-one to the corresponding 3beta-hydroxy compounds. The conversion of 5alpha-cholestan-3-one to 5alpha-cholestan-3beta-ol was only slightly inhibited by the reaction product or by other monohydroxy steroids, but a strong inhibitory effect was noted with cholest-5-en-3-one, 5alpha-cholestane-3beta, 7alpha-diol and 5alpha-cholestan-7-on-3beta-ol. The microsomes, but not high speed supernatant solution, catalyzed the reverse of the cholestanone reductase reaction, namely the conversion of 5alpha-cholestan-3beta-ol to 5alpha-cholestan-3-one in the presence of oxygen and an NADP-generating system. The action of the microsomal preparations upon 5alpha-cholestan-3-one produced 5alpha-cholestan-3alpha-ol in addition to the 3beta-epimer. The 3-alpha-hydroxysteroid dehydrogenase involved functioned with either NADH or NADPH as hydrogen donor. The ratio of 5alpha-cholestan-3beta-ol to 5alpha-cholestan-3alpha-ol formed from 5alpha-cholestan-3-one was approximately 10:1 and was independent of the sex of the animal from which the microsomes were prepared.     

On the mechanism of cerebral accumulation of cholestanol in patients with cerebrotendinous xanthomatosis

The most serious consequence of sterol 27-hydroxylase deficiency in humans [cerebrotendinous xanthomatosis (CTX)] is the development of cholestanol-containing brain xanthomas. The cholestanol in the brain may be derived from the circulation or from 7alpha-hydroxylated intermediates in bile acid synthesis, present at 50- to 250-fold increased levels in plasma. Here, we demonstrate a transfer of 7alpha-hydroxy-4-cholesten-3-one across cultured porcine brain endothelial cells (a model for the blood-brain barrier) that is approximately 100-fold more efficient than the transfer of cholestanol. Furthermore, there was an efficient conversion of 7alpha-hydroxy-4-cholesten-3-one to cholestanol in cultured neuronal and glial cells as well as in monocyte-derived macrophages of human origin. It is concluded that the continuous intracellular production of cholestanol from a bile acid precursor capable of rapidly passing biomembranes, including the blood-brain barrier, is likely to be of major importance for the accumulation of cholestanol in patients with CTX. Such a mechanism also fits well with the observation that treatment with chenodeoxycholic acid, which normalizes the level of the bile acid precursor, results in a reduction of cholestanol-containing xanthomas even in the brain.     

Metabolism of the cholestanol precursor cholesta-4,6-dien-3-one in different tissues

Cerebrotendinous xanthomatosis (CTX) is a lipid storage disease where the basic defect is a lack of the mitochondrial C27-steroid 26-hydroxylase involved in bile acid synthesis (EC 1.14.13.15). Cholestanol and cholesterol accumulate in all tissues. At least part of the accumulation of cholestanol is due to a 7 alpha-dehydroxylation of early bile acid intermediates. Cholesta-4,6-dien-3-one, a proposed intermediate in this pathway, is found in increased concentrations in serum of the patients. This study shows that cholesta-4,6-dien-3-one may be metabolized to 4-cholesten-3-one and cholestanol by liver, adrenals and brain. No conversion was found in intestinal mucosa or in kidneys. The capacity to convert cholesta-4,6-dien-3-one into 4-cholesten-3-one and cholestanol varied in different tissues as well as in different species. The results are discussed in relation to the cholestanol accumulation in CTX.     

Biosynthesis of cholestanol from bile acid intermediates in the rabbit and the rat

Biliary 7 alpha-hydroxy-4-cholesten-3-one (an intermediate in bile acid biosynthesis) may be 7 alpha-dehydroxylated in the gut and further metabolized to cholestanol (Skrede, S., and Bj?rkhem, I. (1982) J. Biol. Chem. 257, 8363-8367). We have now evaluated the quantitative importance of pathway(s) to cholestanol with 7 alpha-hydroxylated C27 steroids as intermediates. After feeding conventionally fed rabbits or rats or germ-free rats with [7 alpha-3H]cholesterol and [4-14C]cholesterol, tissue cholestanol could be isolated with about a 20% lower 3H/14C ratio than present in cholesterol. We conclude that there is a pathway to cholestanol involving 7 alpha-hydroxylated intermediates. Intestinal microorganisms are not essential for this pathway, which accounts for at most 20% of the cholestanol formed in these species. In bile fistula rats, there was also a significant conversion of intraperitoneally injected [7 beta-3H]7 alpha-hydroxycholesterol and [4-14C]7 alpha-hydroxy-4-cholesten-3-one into cholestanol. The enzymes involved in the 7 alpha-hydroxylation/dehydroxylation pathway for the biosynthesis of cholestanol are probably located in the liver. Both 7 alpha-hydroxycholesterol and 7 alpha-hydroxy-4-cholesten-3-one may be intermediates.     

Conversion of cholesterol injected into man to cholestanol via a 3-ketonic intermediate

Cholesterol-3-(3)H,4-(14)C was injected intravenously in man and its transformation to cholestanol was studied. From the (3)H: (14)C ratios in cholestanol isolated from blood, evidence for the participation of a ketonic intermediate in the conversion was obtained. In a second subject given cholestanol-3-(3)H,4-(14)C the (3)H: (14)C ratios in blood sterols remained unchanged for as long as 1 wk after the injection, which showed that cholestanol did not lose tritium by interconversion with cholestanone.     

Isolation of 5 alpha-cholestane-3 beta, 7 alpha-diol from bile of patients with cerebrotendinous xanthomatosis. Inefficiency of this steroid as a precursor to cholestanol

Cerebrotendinous xanthomatosis is a rare, inherited disease characterized by defective bile acid biosynthesis as well as by accumulation of cholesterol and cholestanol. The mechanism behind the accumulation of cholestanol is unknown. Using combined gas chromatography-mass spectrometry, 5 alpha-cholestane-3 beta, 7 alpha-diol could be identified as a minor component in bile from two such patients. There were no significant amounts of this steroid in bile from control subjects. Most probably, the 5 alpha-cholestan-3 beta, 7 alpha-diol found is formed from 7 alpha-hydroxy-4-cholesten-3-one in the liver. 7 alpha-Hydroxy-1-cholesten-3-one, being a normal intermediate in bile acid biosynthesis, is known to accumulate in the liver and bile of patients with cerebrotendinous xanthomatosis, due to a defect of the mitochondrial 26-hydroxylase. The possibility was tested that (7 beta-3H)-labeled 5 alpha-cholestane-3 beta, 7 alpha-diol could be converted into cholestanol by a direct 7 alpha-dehydroxylation in the intestine. This conversion did not occur in rabbits, however, regardless of whether the labelled steroid was administered orally or intracoecally. It is concluded that 5 alpha-cholestane-3 beta, 7 alpha-diol is of little or no importance as a precursor to cholestanol in rabbits. Most probably, this is also the case in patients with cerebrotendinous xanthomatosis.