Desmosterol as the major sterol in L-cell mouse fibroblasts grown in sterol-free culture medium.

The principal sterol synthesized by L-cell mouse fibroblasts is desmosterol. Cholesterol was not detected in these cells when they were grown in a sterol-free culture medium. These findings indicate that, in cells, cholesterol can be replaced by desmosterol. Sterol analyses of six other tissue culture cell lines revealed cholesterol synthesis.

A comparison of the biological properties of androst-5-en-3 beta-ol, a series of (20R)-n-alkylpregn-5-en-3 beta-ols and 21-isopentylcholesterol with those of cholesterol.

The delta 5-sterol, androst-5-en-3 beta-ol, which has no side chain at C-17, did not permit molting of the insect Heliothis zea, growth of either the protozoan Tetrahymena pyriformis, or the yeast Saccharomyces cerevisiae adapted to anaerobic conditions, nor was the sterol esterified by a mammalian microsomal ACAT preparation. However, the sterol did form a liposome with egg lecithin and, when fed to mice, did inhibit hepatic cholesterol synthesis. 21-Isopentylcholesterol also formed a liposome but neither supported the growth of the yeast nor was metabolized by the protozoan. When sterols, 20(R)-n-alkylpregn-5-en-3 beta-ols, with side chains of varying lengths were added to the medium of the protozoan, maximal esterification with fatty acids occurred with the 20(R)-n-pentyl derivative, and maximal inhibition of tetrahymanol formation occurred with the n-butyl, n-pentyl, and n-hexyl derivatives. In all of the assays cholesterol showed a positive response, either permitting molting or growth, being metabolized, inhibiting sterol or tetrahymanol synthesis, or forming a liposome.

The presence of acyl-CoA: cholesterol acyltransferase in Tetrahymena pyriformis W.

1. The esterification of cholesterol was studied in Tetrahymena pyriformis an organism which does not synthesize sterols nor are sterols required for growth. 2. Microsomes catalyzed the esterification of cholesterol in the presence of oleoyl-CoA but not oleic acid or lecithin. 3. The enzyme has a similar sterol substrate specificity to that of mammalian acyl-CoA: cholesterol acyltransferase (ACAT) and was inhibited by the specific ACAT inhibitor 58-035. 4. The enzyme is constitutive since activity was observed in cells grown in sterol-free medium when cholesterol was added to the in vitro assay.

Steric effects at C-20 and C-24 on the metabolism of sterols by Tetrahymena pyriformis.

Cultures of Tetrahymena pyriformis were incubated with various sterols and the extent of dehydrogenation at C-7 and C-22 was determined. The sterols incubated were desmosterol, 22-dehydrodesmosterol, 24-methyldesmosterol, 24 alpha-methylcholesterol (campesterol), 24-methylene-cholesterol, isohalosterol (26,27-bisnorcampesterol, also known as 24,24-dimethylchol-5-en-e beta-ol, a naturally occurring C26-sterol), and 20-isohalosterol. 20-Isohalosterol was not metabolized, while products with delta 7- and delta 22-bonds were formed from isohalosterol and all of the other sterols studied. This confirms an earlier conclusion, based on results with 20-isocholesterol and cholesterol, that inversion of the configuration from 20(R) to 20(S) completely prevents metabolism both in the nucleus and the side chain. On the other hand, changes in the electronics or stereochemistry at C-24 had a direct affect only on metabolism in the side chain. The presence of a methyl group at C-24 reduced the yield of metabolites with a delta 22-bond relative to those with a delta 7-bond producing an accumulation of 7-dehydro metabolite. A double bond at position-24 counteracted this steric effect, presumably by enhancing the rate of dehydrogenation, and a delta 24(28)-bond was more effect than was a delta 24(25)-bond.

Rotational isomerism about the 17(20)-bond of steroids and euphoids as shown by the crystal structures of euphol and tirucallol.

The influence of configuration at C-20 on rotation about the 17(20)-bond in steroids and euphoids was examined by x-ray crystallographic studies of the C-20 epimers euphol and tirucallol. The H atom on C-20 was in back next to C-18 in the crystal structures of both of the compounds, and C-22 was found to be cis-oriented ("left-handed") to C-13 in euphol and trans-oriented to it ("right-handed") in tirucallol. The results, which are consistent with the known left-handed crystal structure of 24(25)-dihydroeuphol and right-handed crystal structure of cholesterol and other natural sterols, lend further credence to the earlier suggestion that rotational isomerism at the 17(20)-bond can arise in C-20 epimers and that there is preference for an arrangement with the 20-H atom adjacent to C-18.

The effects of branching, oxygen, and chain length in the side chain of sterols on their metabolism by Tetrahymena pyriformis.

The ciliated protozoan Tetrahymena pyriformis was incubated with delta 5-sterols which has side chains varying in degree of branching, polarity, and length. Branching at neither end of the side chain was obligatory for metabolism, although removal of C-21 did have a small quantitative effect. Both 21-norcholesterol and sterols lacking a terminal gem-dimethyl group, e.g. 26 (or 27)-norcholesterol, underwent conversion to the corresponding delta 5,7,22-trienols. However, replacement of C-21 with oxygen (20 xi-hydroxy- and 20-keto-21-norcholesterol) was very deleterious. In particular, introduction of a delta 22-bond was abolished, and dehydrogenation in Ring B was strongly reduced. The overall length of the side chain which permitted maximal metabolism corresponded closely to that in cholesterol and other natural sterols. Maximal metabolism was observed only with sterols bearing side chains which had 5 or 6 carbon atoms (other than C-21) attached to C-20. The extent of metabolism fell gradually to zero as the length of the chain was increased or decreased. No metabolism at all occurred with side chains of no carbon atoms (pregn-5-en-3 beta-ol) or as many as 12 carbon atoms (20(R)-n-dodecylpregn-5-en-3 beta-ol) on C-20 other than C-21. Dehydrogenation in the side chain was more sensitive to chain length than was dehydrogenation in Ring B. The data presented here reinforce the view that the enzymes involved in sterol metabolism are actually directed toward binding with sterols. Furthermore, since the sterols become components of the ciliary membrane of this protozoan, the observed structural requirements for metabolism presumably reflect the structural requirements for membrane architecture; and the evidence presented then suggests that the natural length of the sterol side chain is governed by the sterol's membranous function.

Effect of sterol replacement in vivo on the fatty acid composition of Tetrahymena.

The addition of ergosterol to cultures of Tetrahymena pyriformis results in (a) the accumulation of the sterol by the cells; (b) the inhibition of the synthesis of the pentacyclic triterpenoid alcohol, tetrahymanol; (c) the replacement of tetrahymanol by ergosterol in the ciliate membranes. The dry weight and lipid content of sterol-supplemented ciliates did not differ from the controls. Examination of the lipid classes revealed no change in composition except for a higher content of ergosterol in supplemented cells than tetrahymanol in control cultures. The relative proportions of triglycerides, the major classes of polar lipids, 1-alkyl phospholipids and phosphonolipids, appeared unaltered. A complex array of fatty acids is found in this ciliate. Several acids not reported previously in this organism were isolated and identified, and the novel fatty acid 18:2 delta 6, 11, was found in substantial amounts. Ergosterol supplementation altered the proportions of the fatty acids, although not all lipid classes were affected to the same extent. The changes noted were of three general types: (a) a shortening of the fatty acyl chain length in the acids of the normal series; (b) a lowering in the degree of unsaturation; (c) a discrimination between two isomers of lionoleate, 18:2 delta 6, 11 and 18:2 delta 9, 12. The former is elevated in the presence of ergosterol while the latter is depressed. Each class of polar lipids has a distinctive fatty acid composition. Among the glycerophospholipids, cardiolipin and phosphatidylcholine were least affected, while the mixture of 1-alkyl-2-acyl-sn-glycero-3-(2-aminoethyl)-phosphonate and 1,2-diacyl-sn-glycero-3-(2-aminoethyl)-phosphonate was most markedly altered. Sphingolipid fatty acid composition was influenced by ergosterol supplementation. Two changes were noted: (a) a reduction in the length of the hydrocarbon chain; (b) an increase in the proportion of alpha-hydroxy acids. The impact of ergosterol on the fatty acid composition of the polar lipids may be on fatty acid biosynthesis, on incorporation of fatty acids, or on the turnover rates of the fatty acyl groups. Ergosterol is concentrated in the ciliary (limiting) membrane, as are the polar lipids most affected. This localization allows the speculation that the change in fatty acid composition may be related to the maintenance of optimal membrane properties upon the introduction of the sterol.