Unsaponifiable Lipid Research Articles

Identification of the lipid intermediate in yeast mannan biosynthesis.

The unsaponifiable lipid fraction of Saccharomyces cerevisiae contains a phosphorylated component which accepts a mannosyl residue from GDP‐mannose and transfers it to growing mannan chains. Both reactions are catalyzed by membrane‐bound enzymes. The lipid component has been purified more than 1000‐fold (based on lipid‐bound phosphate). After dephosphorylation with an orthophosphoric monoester phosphohydrolase this acceptor was shown by mass spectroscopy to be identical with authentic yeast dolichol. The “lipid” intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol. The “lipid” intermediate intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol monophosphate.Yeast dolichol is a mixture of five homologous polyprenols with saturated α‐isoprene units, as shown by Dunphy et al. in 1967. All these homologues seem also to be present in the cells as monophosphates. As compared to free dolichols the phosphorylated forms only amount to about 10–20%.

An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis.

1. [(14)C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO(3) (-). In a cell-free yeast preparation addition of HCO(3) (-) stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of beta-hydroxy-beta-methylglutaryl-CoA formed from [2-(14)C]- and [1,3-(14)C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-(14)C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-(14)C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.

Phytol and Bacteriochlorophyll Synthesis in Rhodopseudomonas spheroides

Phytol has been separated and identified in the unsaponifiable lipid fraction from wild type Rhodopseudomonas spheroides, but it was not detected in mutant strains blocked at various stages of bacteriochlorophyll synthesis. Incorporation of (14)C-acetate into phytol paralleled bacteriochlorophyll synthesis in suspensions of the wild type incubated anaerobically in the light. The addition of chloramphenicol inhibited both processes. It is concluded that phytol formation is tightly coupled to the synthesis of the pyrrole component of bacteriochlorophyll.

Compartment a1 and semicompartmental approaches for measuring glucose carbon flux to fatty acids and other products in vivo

We have attempted to estimate the flux of glucose carbon to total body fatty acids and to other metabolic end products in Bar Harbor 129/J mice fasted 5-8 hr. Tracer [U-(14)C]glucose was injected intravenously, and the following data were obtained at various times up to 180 min: plasma glucose C specific activity, plasma glucose concentration, total body glycogen, and (14)C in total body fatty acid, total body lipid, unsaponifiable lipid, expired CO(2), and in hepatic and extrahepatic glycogen. The data were analyzed by three techniques, namely, multicompartmental, semicompartmental, and noncompartmental analyses. All three methods yielded comparable rates of glucose C conversion to total body fatty acids (2-3 micro g of glucose C/min/20 g of body weight). Although the semicompartmental approach is extremely simple (it only requires analyses of plasma glucose specific activity as a function of time and (14)C-labeled fatty acid at one point in time), it gives an apparently valid approximation for the flux of glucose C to fatty acids. Other quantitative aspects of glucose metabolism in postabsorptive mice are also considered.

Fluorometric Determination of Vitamin A in Dairy Products

A rapid method has been devised for the determination of vitamin A (retinol) in milk, artificially fortified milk, butter, cheese and ice cream. Samples (1ml or 1g) were saponified and extracted with petroleum ether in stoppered centrifuge tubes. Retinol content of extracts was measured fluorometrically, Chromatography of the unsaponifiable lipids on columns of alumina and hydroxyalkoxypropyl Sephadex demonstrated that retinol was responsible for almost all of the measured fluorescence. Retinol amounts are reported for Canadian whole milk, 2% fat-milk and fortified 2% fat-milk. Samples were obtained monthly between March and November 1971. Mean retinol (μg/100 ml±se) in whole milk was low in March (28.3±3.0) and November (32.1±1.5) and maximal in September (45.8±2.3). A similar cycle was observed in the values for 2% fat-milk which invariably were approximately two-thirds those of whole milk. Sixteen out of 18 fortified milks met their label claims but two had only 19 and 22μg retinol/100ml.

Dolichols, ubiquinones, geranylgeraniol and farnesol as the major metabolites of mevalonate in Phytophthora cactorum.

Farnesol, geranylgeraniol, dolichols and ubiquinones were the main radioactive components of the unsaponifiable lipid recovered from Phytophthora cactorum grown in aerated cultures containing [2-(14)C]mevalonate. The (14)C recovered in each of these components was in the approximate proportion 2:4:3:5. When the culture was not aerated no radioactive ubiquinone was recovered. Most of the (14)C recovered in the dolichols was found in dolichol-15 (37%), with decreasing amounts in dolichol-14 (30%) and -13 (14%) and only a little (5%) in dolichol-16, whereas the major components, by weight, of the mixture (13mug/g of damp-dry tissue) were dolichol-14, -15 and -16 in the approximate proportion of 1:3:1. Radioautography of appropriate chromatograms indicated the presence also of traces of radioactivity in dolichol-9, -10, -11, -12 and -17. Most (80%) of the (14)C recovered in the ubiquinones was associated with ubiquinone-9, the rest being in ubiquinone-8. Most (80%) of the weight of ubiquinones (19mug/g of damp-dry tissue) was also ubiquinone-9. The identification of these compounds was by chromatographic methods and, for the ubiquinones and dolichols, was confirmed by mass spectrometry. In addition, the incorporation of 4R- and/or 4S-(3)H from [4-(3)H]-mevalonates showed the expected stereochemistry of biosynthesis, namely that farnesol, geranylgeraniol and ubiquinones were biogenetically all trans and the dolichols each contained three biogenetically trans isoprene residues, the remaining residues being biogenetically cis. The distribution of (14)C in the components of the whole lipid of the fungus was consistent with 97% of both the farnesol and geranylgeraniol being present as the fatty acid ester. The corresponding value for dolichols was 37%. The observation by other workers, that this fungus does not form either squalene or sterol, was confirmed.

The identification of ceramides and glyceryl ethers in unsaponifiable lipid of human aorta

Unsaponifiable material from a lipid fraction isolated from atherosclerotic human aorta, contained cerebrosides, which had been previously identified, ceramides, and glycerol ethers, as well as other, still unidentified, substances. Identification of the ceramides and glycerol ethers was made primarily by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. Ceramides were present with the fatty acids 16:0, 23:0, 24:0, and 24:1. Other fatty acids were found which were probably primarily associated with ceramides. These acids were 18:0, 18:1, 20:0, 22:0, and 25:0. The glycerol ethers were 1-glycerol ethers of the fatty alcohols 16:0, 18:0, and 18:1. Glycerol ethers may well have been formed from diacylglycerol ethers during the isolation procedure.

Steroids in the pineal gland of the domestic fowl ( Gallus domesticus): Cholesterol and its biosynthesis

1. 1. Cholesterol was identified as a constituent of the pineal gland of the domestic fowl using thin-layer and gas-liquid chromatography; the pineals of laying hens had a higher cholesterol content (0·55 per cent) than those from young birds (0·08 per cent). 2. 2. Pineal tissue from young birds incorporated radioactivity from [2- 14C]- dl-mevalonic acid lactone or sodium [1- 14C]-acetate in vitro into the unsaponifiable lipids in variable amounts; the former precursor was used preferentially. 3. 3. Chromatographic analysis of the lipids showed that radioactivity was associated with the “cholesterol”, “lanosterol” and “squalene” fractions in proportions varying according to the precursor. 4. 4. Only squalene was identified unambiguously as a biosynthetic product of the incubation of pineals with [2- 14C]-mevalonate. Neither the “cholesterol” nor the “lanosterol” fractions gave derivatives which recrystallized to constant specific activity. 5. 5. Comparable incubations of adrenal tissue from a young bird using the same radioactive precursors afforded similar results.

Comparative metabolism of mevalonate in the intestinal mucosa of rat, cat, and dog

1. 1. Mucosal scraping of the dog and the cat but not the rat intestine gives stable preparations that metabolize mevalonate into unsaponifiable lipids. 2. 2. The activity of the rat mucosa could not be restored by thiols. 3. 3. However, washing the intestine with Krebs-Ringer-phosphate medium and subsequent incubation with 0.1 M potassium phosphate buffer, pH 7.4, supplemented with 8 × 10 −3 M calcium chloride, 7 × 10 −3 M magnesium sulphate, and 5.6 × 10 −3 M glucose, preserves the activity of the mucosa of the rat intestine. 4. 4. Bile-salts inhibit mevalonate metabolism both at the site of squalene synthesis as well as the conversion of squalene to sterols.

Lipids in brain tumors.

ELATIVELY few studies have been performed on lipids of brain tumors. The pioneer study of Brante ~ showed that brain tumors in general have a lipid content lower than normal brain and contain esterified cholesterol, which is absent from normal adult brain tissue. Phospholipids are also present in small amounts/The presence in brain tumors of free fatty acids, triglycerides, and cholesterol esters, and certain lipids absent from normal brain, has been detected in our laboratory/Azarnoff and coworkers t demonstrated that human glioblastomas, astrocytomas, and other intracranial tumors are capable of incorporating 14C-acetate into cholesterol in contrast with normal brain tissue which is known to synthesize cholesterol only to a limited extent. Paoletti, et al./ demonstrated that the Zimmerman ependymoma can convert various precursors into cholesterol much more efficiently than normal brain of the same animals. A typical feature of brain tumors is the presence of desmosterol (24-dehydrocholesterol), which differs from cholesterol only by an extra double bond in the lateral chain. This sterol is the last precursor of cholesterol in one of its biosynthetic possible routes. However, in the liver desmosterol does not accumulate, despite high levels of cholesterol synthesis; in brain during maturation, however, large amounts of desmosterol are found as well as in human glial tumors? ,~ In recent experiments, large quantities of desmosterol were detected in brain tumors of the rat induced in our laboratories by nitrosourea derivatives according to Druckrey, et al? In ethylnitrosourea-induced oligodendrogliomas, desmosterol accounted for 10% to 15% of the total sterols. 12 Desmosterol was considerably higher in some neurinomas, which is not surprising since these tumors are quite malignant and fast-growing when compared with human neurinomas. The presence of consistent pools of desmosterol in brain tumors is connected with both the high sterol biosynthetic rate and with a blockade of desmosterol transformation into cholesterol. The ability of human brain tumors to synthesize both desmosterol and cholesterol from mevalonate was checked in vitro and compared with that of normal brain tissue. The results G indicate that the incorporation of mevalonate in the unsaponifiable lipid fraction is very low for the slow-growing neurinomas, which do not synthesize cholesterol, while the mest notable difference between glial tumors and normal brain is at the sterol level only. In fact, normal brain incorporated less radioactivity into sterols, and most of it was detected in cholesterol. The reverse was found in glial tumors, which showed a high sterol synthesis, and the ratio between synthesized desmosterol/cholesterol was higher in the glioblastomas than in other less malignant gliomas. Comparable results were obtained by incubating in vitro an experimental ependymoblastoma, which, after 1 hour of incubation, contained a twofold increase of radioactivity in desmosterol than in cholesterol. Desmosterol accumulation in brain tumors may be magnified by Triparanol administration. This drug, inhibiting desmosterol reductase, induces accumulation of desmosterol in blood but not in mature brain. This was demonstrated in mice with experimental

Utilization of Dietary Amino Acids in Lipid Synthesis by Aseptically Reared Musca domestica1,2,3

Pupae obtained from house fly larvae ( Musca domestica L.) reared on aseptic synthetic diets containing alanine-U-14C, phenylalanine-U-14C, glutamate-U-14C, leucine-U-14C, or methionine-G-3H contained radioactivity in both fatty acids (84–95%) and unsaponifiable lipids (5–16%). As a lipid precursor, leucine and glutamate were the best and methionine, alanine, and phenylalanine the least utilized. Fatty acids found were myristic (1.7%), palmitic (28.7%), palmitoleic (39.7%), stearic (trace), and oleic (29.9%). Distribution of radioactivity in unsaponifiable lipids were mainly hydrocarbons (73%). A new biochemical difference between the house fly and other animals is reported, and their ability to convert amino acids into lipids and energy production is discussed.

Investigations of the Capacity of Synthesizing 3β-Sterols in Mollusca Vii. the Biosynthesis and Composition of 3β-Sterols in the Neogastropods Buccinum Undatum and Neptunea Antiqua

Buccinum undatum and Neptunea antiqua are able to synthesize saponifiable and unsaponifiable lipids from both sodium acetate-1- 14 C and DL-mevalonate-2- 14 C (DBED salt). Buccinum undatum did synthesize squalene from acetate as well as from DL-mevalonic acid-2- 14 C. Buccinum is able to synthesize 3β-sterols from mevalonate. Both species showed a low incorporation of radioactivity into lipids. This phenomenon seems to be a common property of carnivorous molluscs. The sterol composition of the animals was determined by means of gas-liquid chromatography. In both animals cholesterol was the principal sterol. The sterol composition of these Buccinidae showed strong resemblance to that of the Muricidae.

The cell-wall lipids of cells of tetracycline-sensitive and tetracycline-resistant strains of streptococcus pyogenes

1. 1. A comparison of the total solvent-extractable cell-wall lipid and surface lipid of a range of laboratory and recently isolated strains of Streptococcus pyogenes has been made. 2. 2. Strains of S. pyogenes which are sensitive to tetracycline have 4–6% (w/w) cell-wall lipid and no detectable surface lipid. Strains resistant to tetracycline have up to 3 times as much extractable lipid and also possess a considerable amount of surface lipid. 3. 3. Naturally occurring, tetracycline-resistant cells have a greater proportion of unsaponifiable lipid than the corresponding tetracycline-sensitive cells. 4. 4. Tetracycline-sensitive cells isolated before about 1935 and subcultured for a long period have a smaller proportion of saponifiable lipid than recently isolated strains.

Sterols in red algae (Rhodophyceae): Variation in the desmosterol content of dulse ( Rhodymenia palmata)

1. 1. Desmosterol ranged from 30·6 to 97·2 per cent and averaged 73·6 per cent of the total sterol in twenty-three samples of dulse collected during the months of June–November. 2. 2. Cholesterol was the second principal sterol ranging from 2·1 to 49·2 per cent and averaging 13·0 per cent. 3. 3. Twelve minor sterols were detected in various samples and seven were identified by GLC. 4. 4. The sterol compositions were variable and did not depend on the location or time of collection. 5. 5. Average total lipid, unsaponifiable lipid and sterol contents were highest in samples taken during June, July and August. Sterol content ranged from 0·005 to 0·019 per cent and averaged 0·012 per cent of the dry weight of dulse. 6. 6. Cholesterol was the principal sterol in three other red algae, Ptilota serrata, Furcellaria fastigiata and Phyllophora membranifolia.

The characterization of undecaprenol of Lactobacillus plantarum.

Evidence for the presence of undecaprenol in the unsaponifiable lipid of Lactobacillus plantarum (N.C.I.B. 6376) is presented. Characterization of the compound was based mainly on mass, i.r. and n.m.r. spectrometry. The prenol was isolated at a concentration of 40mug/g wet wt. of bacteria and contained over 90% (1.0-5.4% of the dose) of the (14)C present in the unsaponifiable lipid after incubation of the bacteria with [2-(14)C]mevalonate. N.m.r. spectrometry indicated the presence of two internal trans-, one alpha-cis- and seven internal cis-isoprene residues per molecule. The (3)H/(14)C ratios of the prenol after incubation of the bacteria with [2-(14)C,(4R)-4-(3)H(1)]- and [2-(14)C,(4S)-4-(3)H(1)]-mevalonate were in agreement with this stereochemistry. There was no evidence of saturated isoprene residues in the molecule. The undecaprenol appeared to be accompanied by much smaller quantities of decaprenol and nonaprenol.

The liquid spectroradiochromatograph—an automated method for the simultaneous separation, identification, purification, estimation and radioassay of the components of a mixture of unsaponifiable lipids

An automated system which we have termed the Liquid Spectroradiochromatograph, capable of monitoring the effluent from a liquid chromatography column for solute concentration, radioactivity and ultraviolet and visible light absorption, is described. Its usefulness in the analysis of mixtures of unsaponifiable lipids is discussed.

Inhibition of sterol synthesis in vitro by metabolites of phenylalanine

Studies with homogenates and 10000 × g supernatants of brain and liver demonstrated that phenylpyruvate, phenyllactate and phenylacetate markedly inhibited the incorporation of [ 14C]mevalonate into unsaponifiable lipids and digitonin-precipitable sterols. Phenylpyruvate was the most potent of the inhibitory compounds. The deaminated derivatives of phenylalanine blocked the decarboxylation of mevalonic acid pyrophosphate, preventing the formation of light petroleum-soluble polyprenols. In contrast, phenylalanine which also inhibited sterol synthesis in vitro seemed to inhibit at some step beyond the formation of polyprenols.

The Conversion of Cholesterol to Δ5,7,22-Cholestatrien-3β-ol by Tetrahymena pyriformis

The protozoan Tetrahymena pyriformis was incubated in a growth medium containing added unlabeled cholesterol or 26-14C-cholesterol with the following consequences: (a) the biosynthesis of tetrahymanol, a pentacyclic triterpenoid alcohol produced in the organism by squalene cyclization, was inhibited, and (b) the cholesterol was accumulated by the cells and converted metabolically to a mixture of other sterols containing Δ5,7,22-cholestatrien-3β-ol(7,22-bisdehydrocholesterol) as the major component, together with 22-dehydrocholesterol and possibly a small amount of 7-dehydrocholesterol. These three sterols, along with unconverted cholesterol, were obtained in the unsaponifiable lipid fraction from the cells, and were separated from one another by silicic acid-silver nitrate column chromatography of the corresponding acetylated derivatives. The 22-dehydrocholesterol and the recovered cholesterol were rigorously identified by comparisons of various chromatographic, spectral, and other properties of the alcohols and their acetates with those of authentic samples. The tentative identification of the 7-dehydrocholesterol was based on chromatographic comparisons of the acetate derivative with authentic material. The structure of the 7,22-bisdehydrocholesterol, a previously unknown compound, was established on the basis of ultraviolet, infrared, nuclear magnetic resonance, and mass spectral data for both the free sterol and its acetate derivative, and also by chemical conversions of the acetate to known compounds. Thus, hydrogenation of the 7,22-bisdehydrocholesteryl acetate with a Raney nickel catalyst gave lathosteryl acetate, and periodate-permanganate oxidation of the triene acetate gave isovaleric acid. It is suggested that the conversion of cholesterol to 7,22-bisdehydrocholesterol in the cells proceeds by way of either 7-dehydrocholesterol or 22-dehydrocholesterol as intermediate.

Hydrocarbons and alcohols of basking shark and pig liver lipids

AbstractFour samples of the unsaponifiables of basking shark liver oil were adsorbed on alumina and eluted to yield Fractions 1–5, inclusive. Analyses by temperature programmed GC and by silica gel chromatography showed hydrocarbons in the first four fractions with squalene increasing to Fraction 3 and the pristane level being highest in Fraction 1. Aside from pristane and squalene, other hydrocarbons occurred at levels of 420–750 mg% in the oils on a weight basis, of which about 60% constituted a series of n‐paraffins (relative carbon number range: 15.0–38.0) together with smaller amounts of at least one branched chain saturated group. Unsaturated hydrocarbons eluted mainly after squalene. The oils contained up to 460 mg% sterol and 78–270 mg% alcohols of C10 to C30, the ratio of saturated to unsaturated members being about 1.6. The composition of the unsaponifiable lipids of pig liver was quite different from that of the marine oils. It contained 10.6% sterol in addition to 400 mg% alcohols, the latter consisting of 81.8% saturated components (C12 to C31; ratio of saturated: unsaturated members, 4.4). The hydrocarbons comprised 450–700 mg% of the unsaponifiable mixture and squalene, paraffins and additional unsaturated components occurred at levels of 20.6, 24.4 and 11.9 mg%, respectively. The saturated hydrocarbons were high in normal homologs of relative carbon number range, 15 to 36; pristane could not be detected.

A study on the synthesi and absorption of cholesterol in hydatid cysts ( Echinococcus granulosus)

1. 1. Fresh scolices of Echinococcus granulosus failed to incorporate the label from sodium acetate-1-C 14 or mevalonic acid lactone-2-C 14 into squalene or cholesterol. However, some radioactivity was detected in the unsaponifiable lipid. This warm attributed to an intermediates(s) formed before squalene in the cholesterol biosynthetic pathway. 2. 2. In vivo experiments showed that cholesterol passes freely from the host tissues to the hydatid cysts.