Fatty acid and sterol specificity in the biosynthesis of steryl esters by enzyme preparations from spinach leaves

Acetone powders prepared from the 20,000g participate fraction of spinach (Spinacia oleracea L.) leaves catalyzed the formation of steryl esters from free sterol and 1,2-diacylglycerol as the acyl donor. There was no sterol specificity when cholesterol, sitosterol, and campesterol were compared. When rates of sterol ester biosynthesis were compared using different 1,2-diacylglycerols it was found that the shorter chain fatty acids and the more unsaturated fatty acids were preferred. When the substrate concentration of diacylglycerol was varied, the maximal velocities obtained with the different substrates were dipalmitoleoyl- >dilinolenoyl- >dioleoyl- >dilinoleoyl-glycerol. It was demonstrated by silver nitrate thin-layer chromatography that the fatty acids of the supplied diacylglycerols were transferred to the sterol. When diacylglycerol mixtures were supplied, it was found that unsaturated diacylglycerols greatly stimulated conversion of saturated diacylglycerols to saturated steryl esters. For an equimolar mixture of dipalmitoyl-, dioleoyl-, dilinoleoyl-, and dilinolenoyl-glycerol, about equal amounts of the four steryl ester species were synthesized.     

Content and Fatty Acid Composition of Steryl and Wax Esters in Germinating Spores of Polytrichum commune

Polytrichum commune spores contained 5.61 ± 0.52 mg steryl and wax esters, including volatile compounds, per 100 mg dry weight of spores. Volatile compounds were not found in 3-h-old sporelings. The content of the steryl and wax ester fraction, excluding the volatile compounds, is slightly increased during the first 6 h of germination. Thereafter, the content is decreased throughout the germination. Thus, 3-day-old sporelings contained 0.52 ± 0.05 mg steryl and wax esters per 100 mg dry weight of spores. In connection with protonema growth, steryl and wax esters were produced, and the 7-day-old cultures contained 5.09 ± 0.37 mg steryl and wax esters per 100 mg dry weight of spores. The main fatty acids of the steryl and wax ester fraction of dry spores and germinating spores as well as of protonemata were palmitic, oleic, linoleic and linolenic acids. Polyunsaturated C 20 acids were present only in trace or small amounts. Phytanic and phytenic acids were found in small amounts in dry spores, in 3- to 72-h-old sporelings, and in protonemata.     

Common bile duct obstruction caused by the hydatid daughter cysts

Echinococcosis is a human parasitary disease. In 2002, 29 new cases of liver echinococcosis were recorded in Croatia. Liver is the most common site of hydatid cysts. Nine patients with echinoccocal liver disease were operated in our department in 2002. Here we present a case where a patient with verified hydatid cyst in the left liver lobe developed high fever, jaundice, nausea, vomiting and pain in the upper abdomen. The symptoms were initially ascribed to the acute cholangitis. After unsuccessful antibiotic treatment, computerized tomography and endoscopic retrograde cholangiopancreatography (ERCP) were performed, demonstrating daughter cysts in the common bile duct. During ERCP, papilotomy was made and daughter cysts were extracted. Hydatid cyst was surgically removed, and a communication between the cyst and left hepatic duct was noted during surgery. Pericystectomy, choledochotomy, removal of remaining daughter cysts from the common bile duct, and sutures of left hepatic duct were performed. The patient recovered fully after the surgery. One of the possible complications of the liver hydatid cysts is the communication between cyst and the biliary tree. Such communications are usually asymptomatic, but symptoms can also mimic acute cholangitis and jaundice, which may lead to the misdiagnosis of the patient's condition.     

Synthesis of lipids from acetate by human preputial and abdominal skin in vitro

Lipogenesis in vitro from acetate-1-(14)C was studied in human preputial skin and abdominal skin. Radioactive lipids were separated by column chromatography on Florisil and by thin-layer chromatography on silica gel. Radioactivity was incorporated chiefly into the triglyceride, sterol, and polar lipid fractions, while lesser amounts of (14)C were found in the hydrocarbon, wax, diglyceride, monoglyceride, and fatty acid fractions; labeling of steryl esters was minimal. On thin-layer chromatography, the radioactive polar lipids had mobilities similar to lysolecithin, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidic acid. The radioactive fatty acids of the different lipid fractions were separated by gas-liquid chromatography. The major (14)C-labeled acids were 16:0 and 18:0. Radioactivity was also detected in acids 14:0, 15:0, 16:1, 18:1, 18:2, 20:0, 20:1, 22:0, 24:0, 24:1, and 26:0. No radioactivity could be detected in arachidonic acid, although this fatty acid comprises 9% of the chromatographed fatty acids. The pattern of incorporated (14)C was different from the percentage mass composition of the fatty acids. Skin is therefore active in the biosynthesis of a wider variety of lipids than previously demonstrated.     

Distribution of arachidonic and eicosapentaenoic acids in the lipids of mosses.

Lipid classes from four species of mosses, Mnium cuspidatum, and Mnium medium from Minnesota, and Hylocomium splendens and Pleurozium schreberi from Alaska, were analyzed. The total lipids of all species contained 30-40% arachidonic and eicosapentaenoic acids. However, the lipids from the Alaskan mosses contained about 75% neutral lipids (triacylglycerols, steryl esters and wax esters) whereas the lipids of the other species contained only 20% or less of these neutral lipids. Consistently, monogalactosyldiacylglycerols and phosphatidylethanol-amines were enriched in arachidonic acid and the galactolipids in eicosapentaenoic acid. The distribution of these acids in the phospholipids shows some preference for position 2. Together, the highly unsaturated C20 acids represented 80% of acyl groups in steryl esters. In triacylglycerols they were at average levels, while they were much less in sulfolipids and phosphatidylglycerols. Wax esters contained very little of the highly unsaturated acids but appreciable amounts of phytol and phytenic acid were found as wax constituents.     

Sterols in hardening winter wheat

The main sterols in winter wheat crowns and roots were sitosterol and campesterol, with significant amounts of stigmasterol and traces of cholesterol. The main groups of sterol-containing lipids were free sterols, steryl glucosides, steryl esters and esterified steryl glucosides. Sterol analysis within each group showed little difference between them. Steryl esters were relatively rich in cholesterol and poor in stigmasterol. Free sterols were rich in stigmasterol. Low temperature caused an increase in sterol content but had little effect on sterol composition and sterol to lipid P ratio. There was some increase in steryl esters and some decrease in free sterols. Cholesterol and stigmasterol decreased in the steryl ester and free sterol fractions, respectively. There was little evidence for involvement of sterols in winter wheat frost hardening.     

Effect of prolactin on the phospholipid composition and cholesterol level in liver microsomes of the carp during acclimation to different temperatures

The phospholipid composition, content of cholesterol and its esters in the carp (Cyprinus carpio L.) liver microsomes depend on the environmental temperature. The free cholesterol amount and cholesterol/phospholipids ratio in microsomes decrease after the lowering of temperature from 20 to 5 degrees C. The temperature elevation to 30 degrees C results in an increase of the cholesterol ester content. The relative proportions of phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol, phosphatidyl serine, phosphatidic acid increase with a significant decrease of the unidentified phospholipids amount at 30 degrees C. Prolactin affects the cholesterol content and phospholipid composition of liver microsomes. The hormone has a more pronounced effect at subextremal temperatures (5 and 30 degrees C). The actions of prolactin and temperature on the cholesterol content are similar. The hormone influence on the membrane phospholipid composition is opposite to the effect of the temperature acclimation. The possible role of prolactin in the temperature adaptation of the membrane lipids metabolism in poikilotherms is discussed.     

Partial characterization of steryl ester biosynthesis in spinach leaves

Acetone powders of a 20,000g pellet fraction from spinach leaves (Spinacia oleracea L.) synthesized [4-(14)C]cholesteryl esters when incubated with [4-(14)C]cholesterol. The reaction was inhibited by digitonin. There was a reciprocal relationship between the decline of label in cholesterol and its incorporation into cholesteryl ester, indicating that free cholesterol was the direct precursor for cholesteryl ester biosynthesis. The hydrolysis of cholesteryl [1-(14)C]palmitate into free cholesterol and [1-(14)C]palmitate was not detected in these acetone powder preparations. Exogenous cholesteryl palmitate had no effect on the esterification of [4-(14)C]cholesterol. The data indicate that an esterase-type mechanism was not involved in the biosynthesis of these steryl esters. Label from [1-(14)C]palmitoyl-CoA was incorporated into steryl esters when incubated with spinach leaf acetone powder preparations. The optimal buffer for steryl ester biosynthesis was 2-(N-morpholino)ethanesulfonate and the optimal pH was 6. Iodoacetamide, N-ethylmaleimide, and dithiothreitol had no effect on the esterification reaction. Ethylenediaminetetraacetate, MgCl(2), CaCl(2), MnCl(2), and ZnSO(4) inhibited at concentrations of 10 to 30 mm.     

The lipids and phospholipids of hydatid protoscolices of Echinococcus granulosus (cestoda)

Frayha G. J., Bahr G. M. and Haddad R. 1980. The lipids and phospholipids of hydatid protoscolices of Echinococcus granulosus (Cestoda). International Journal for Parasitology10: 213–216. The protoscolices of Echinococcus granulosus were isolated from hydatid cysts. The phospho- and neutral lipids of the protoscolices were determined. The seven major classes of lipids namely, phospholipids, fatty acids, mono-, di- and triglycerides, cholesterol and cholesterol esters were identified and quantitatively determined. The phospholipids isolated were cephalin, lecithin, lysolecithin, phosphatidyl inositol, phosphatidyl serine and sphingomyelin. Lecithin and cephalin were the most abundant. The signiflcance of this finding is discussed.     

The Neutral Lipids of Paramecium tetraurelia: Changes with Culture Age and the Detection of Steryl Esters in Ciliary Membranes1

Neutral lipids, particularly triglycerides, accounted for the major decrease in the total lipid content in Paramecium cells that occurs with culture age. Sterols, triglycerides, and steryl esters were the major classes of neutral lipids in cells and isolated cilia. Free as well as high concentrations of esterified sterols were detected in purified ciliary membrane preparations. Stigmasterol and 7-dehydrostigmasterol were the major components of both free and esterified sterols of cells and cilia; however, when cholesterol was present in the growth medium, it was desaturated to 7-dehydrocholesterol and incorporated into cellular and ciliary lipids. Free fatty acids from cells and triglycerides from cells and cilia were low in polyunsaturated fatty acids and reflected the composition of fatty acids in the culture medium. An exception was the reduced concentration of stearate in triglycerides from whole cells. Greater than 50% of triglyceride fatty acids from cilia were saturated. The fatty acid compositions of cellular triglycerides and ciliary steryl esters did not change with culture age, but those of cellular steryl esters and ciliary triglycerides did change. In comparison with phospholipids, these neutral lipid fatty acid compositional changes were smaller. The sensitivity of these stigmasterol-containing cells to polyene antibiotics indicated that they were killed by nystatin > filipin > amphotericin B. The unexpected finding of high concentrations of steryl esters in ciliary membrane preparations is discussed.     

Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

Lipid metabolism in chromoplast membranes from the daffodil: Glycosylation and acylation

The non-photosynthetic chromoplast membranes from the corona ofNarcissus pseudonarcissus L. were investigated for their lipid synthetic capabilities. The following activities were detected: galactosylation of diacylglycerol and galactosydiacylglycerols, glycosylation of sterols, acylation of monogalactosyldiacylglycerol and steryl glycosides from an unknown endogenous donor, acylation of phospholipids from acyl-CoA, and acylation of phosphatidyl inositol from phosphatidyl choline. Furthermore, activities of an acyl thioesterase, a sugar epimerase, and a phospholipase A₂ were measured.Abbreviations MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol - TGDG tri-and tetragalactosyldiacylglycerol - SG steryl glycoside - SL sulfolipid - ACP acyl carrier protein     

Incorporation of [4-14C] cholesterol into steryl derivatives and saponins of oat (Avena sativa L.) plants

Sterols from both green and etiolated oat plants (Avena sativa) contain sitosterol, stigmasterol, cholesterol, ⁷-stigmastenol, ⁷-cholestenol and campesterol. In the saponin fraction avenacosides A and B and 26-desgluco-avenacosides A and B were detected. Etiolated plants incorporated [4-¹⁴C] cholesterol into steryl derivatives (esters, glycosides and acylated glycosides) and also into all of the 4 saponins. [4-¹⁴C] sitosterol, however, is incorporated only into steryl derivatives, but not in saponins. From this it is concluded that cholesterol, but not sitosterol is the in vivo precursor of oat saponins.Abbreviations GLC gas liquid chromatography - I.D. inner diameter - TMS trimethylsilyl - dpm decompositions per minute     

Elevation of liver diacylglycerols and molecular species of diacylglycerols in rats fed a lipogenic diet

The concentration of diacylglycerols in rat liver was measured by a newly developed procedure that entailed a) freeze-clamping of the liver in situ, b) lipid extraction with dimyristoylglycerol as an internal standard, c) thin-layer chromatography, and d) gas-liquid chromatography. Molecular species of diacylglycerol and total diacylglycerols were quantified. The average level of diacylglycerol in livers of chow-fed rats was 102 micrograms/g wet wt, equivalent to about 180 nmol/g wet wt. A high-carbohydrate fat-free diet, known to increase the rate of fatty acid synthesis, greatly increased the liver diacylglycerol concentration relative to the level observed in rats fed laboratory chow. Diacylglycerol molecular species that contained 16-carbon fatty acids were most markedly elevated. Liver triacylglycerol, free cholesterol, and esterified cholesterol were concurrently increased. Molecular species of triacylglycerols and cholesteryl esters that contained 16-carbon fatty acids were elevated to the greatest degree. The concentrations of total triacylglycerol and cholesteryl esters exhibited a high correlation in the livers of all animals studied, suggesting their coexistence in metabolic pools, predominantly the cores of lipid droplets and newly assembled very low density lipoprotein particles. The correlation of liver diacylglycerol and triacylglycerol contents in the chow-fed rats suggests that the diacylglycerol concentration may be a rate-determining factor in triacylglycerol synthesis when diacylglycerol levels are in the observed range of 70-150 micrograms/g wet wt. In conclusion, when the rates of fatty acid synthesis and hence triacylglycerol synthesis are increased in the liver, the steady state concentrations of diacylglycerols are also elevated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Separation and analysis of steryl and wax esters from Dicranum elongatum

Abstract Complete separation of the steryl and wax esters in the subarctic moss Dicranum elongatum was achieved on MgO thin-layer plates without any notable alteration of the acyl and alkyl moieties of the esters. Gas chromatography-mass spectrometry of the hydrolyzed fraction showed that the sterols (campesterol, stigmasterol, sitosterol, cycloartenol, 24-methylene cycloartanol and an unidentified sterol) were primarily esterified with unsaturated fatty acids 18:2 ω 6, 18:3 ω 3 and 20:4 ω 6. In contrast, the wax alcohols (l-octadecanol, phytol and geranylgeraniol) were mainly esterified with saturated fatty acids with 16:0, 18:0 and 20:0 as major components. No great differences were found in the fatty acid pattern of the steryl esters between different portions of the shoot. Slight differences, however, were found in the proportions of ω 3 and ω 6 fatty acids. In the wax esters a clear decrease was found in the proportions of 18:0 and 20:0 acids with increased shoot age accompanied by a slight increase in the proportions of 14:0, 20:4 ω 6 and phytenic acid.     

Effect of acute hypobaric hypoxia on 32-P incorporation into phospholipids of alveolar surfactant, lung, liver and plasma of rat.

Exposure of adult rats to hypobaric hypoxia caused hypolipidemia, hypotriglyceridemia and hypophospholipidemia. Hypobaric hypoxia produced an increase in liver triglyceride and cholesterol levels and a decrease in lung triglyceride, total phospholipid and phosphatidyl choline. The proportion of phosphatidyl choline in the pulmonary surfactant fraction I phospholipids (responsible for reducing surface tension) decreased (55.2% as compared to 80.4% in control animals). Incorporation of 32-P into liver phosphatidyl ethanolamine was significantly increased, incorporation into lung phosphatidyl choline and phosphatidyl ethanolamine was increased whereas a decreased incorporation into plasma phosphatidyl choline was observed. The data suggest an enhanced lipid synthesis in liver with a probable impairment of mobilization into plasma.     

Glycolipid composition of Hevea brasiliensis latex

Glycolipids of fresh latex from three clones of Hevea brasiliensis were characterized and quantified by HPLC/ESI-MS. Their fatty acyl and sterol components were further confirmed by GC/MS after saponification. The four detected glycolipid classes were steryl glucosides (SG), esterified steryl glucosides (ESG), monogalactosyl diacylglycerols (MGDG) and digalactosyl diacylglycerols (DGDG). Sterols in SG, ESG and total latex unsaponifiable were stigmasterol, β-sitosterol and Δ⁵-avenasterol. The latter was found instead of fucosterol formerly described. Galactolipids were mainly DGDG and had a fatty acid composition different from that of plant leaves as they contained less than 5% C18:3. Glycolipids, which represented 27–37% of total lipids, displayed important clonal variations in the proportions of the different fatty acids. ESG, MGDG and DGDG from clone PB235 differed notably by their higher content in furan fatty acid, which accounted for more than 40% of total fatty acids. Clonal variation was also observed in the relative proportions of glycolipid classes except MGDG (8%), with 43–51% DGDG, 30–34% SG and 7–19% ESG. When compared with other plant cell content, the unusual glycolipid composition of H. brasiliensis latex may be linked to the peculiar nature of this specialized cytoplasm expelled from laticiferous system, especially in terms of functional and structural properties.     

The surface wax composition of the exuviae and adults of Aleyrodes singularis

Long-chain aldehydes, alcohols, hydrocarbons and wax esters were major components of the external lipids of adult Aleyrodes singularis. In exuviae, acetate esters replaced the hydrocarbons as a major component. The major long-chain alcohol and aldehyde from adults were C32 and were essentially the exclusive components of the wax particles. The major alcohol from exuviae was C26 and the aldehydes were C26, C28, C30 and C32. The major acetate esters were C28 and C30 in both adults and exuviae. There were wax esters of similar carbon number in adults and exuviae although the exuviae had a greater amount of wax esters with unsaturated fatty acids. The fatty acid and alcohol composition of the wax esters differed markedly between adults and exuviae. Wax esters of adults had similar amounts of C16, C18, C20, C22 and C24 fatty acids while those from exuviae contained largely C16 and C18. The major alcohol in the wax esters of adults was C22 and those of exuviae were C26 and C28. The distribution of fatty acids and alcohols among wax esters of varying chain length also differed between adults and exuviae: in adults C22 was the major fatty acid found in the dominant wax ester, C44 and the C22 alcohol was the major alcohol and found in wax esters C42 and C44. In exuviae C16 and C18 were the major fatty acids found in most wax esters and a C28 alcohol was the major alcohol found in wax esters C44 and C46, the two dominant wax esters in exuviae. It was clear that the difference in chemistry of the wax esters between the adults and exuviae is not evident unless the acid and alcohol moieties are characterized.     

Studies on furan fatty acids of salmon roe phospholipids

Mature salmon roe lipids were found to consist of triacylglycerols (63%), phospholipids (30%), sterols (4.2%), steryl esters (0.7%), and other minor components. In the steryl esters and phospholipids, furan fatty acids were detected instead of the triacylglycerols of the testes lipids in male fish. The representative 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid (F6) amounted to 3.8% and 0.6% of the total fatty acids in each fraction, respectively. However, the absolute amount of the acid in the phospholipid was much more than that contained in the steryl esters. The characteristic distribution of the furan acids found in the phospholipids was common to the steryl esters in the liver. Large amounts of furan acids were contained in phosphatidylcholine (PC) rather than in phosphatidylethanolamine. For positional analysis of furan fatty acids in PC, furan-containing species in the molecule were concentrated fourteenfold by using selective hydrogenation and repeated silica gel column chromatography. A series of furan fatty acids in PC was found to be exclusively linked to the sn-1 position. The amount of the acids in the roe exclusively linked to the sn-1 position. The amount of the acids in the roe phospholipids was comparable with that in the testes triacylglycerols. The physiological roles of furan fatty acids are discussed.     

Sterols in Germinating Seeds and Developing seedlings of Longleaf Pine, Pinus palustris

Sterols in germinating embryos and young seedlings of longleaf pine (Pinus palustris Mill.) were identified and quantities determined for different periods after germination. Sterol analyses were performed by gas-liquid chromatography (GLC) and verified by combination of GLC-mass spectrometry. Campesterol and β-sitosterol were two major sterols which accounted for most of the sterol composition while stigmasterol was present in very small amounts. No cholesterol was revealed by GLC-mass spectrometry although there was a minor peak appearing on the sterol gas-liquid chromatograms with a retention time close to that of authentic cholesterol. By fractionation, three different forms of sterols were obtained: steryl esters, steryl glycosides, and free sterols. The sterols were mainly found in the esterified fraction, while steryl glycosides and free sterols only made up a small portion of the total sterol value. The total sterol content in general increased during seedling development, and this increase reflected mainly a change in steryl esters. The low levels of both free and glycosidic sterols remained nearly unchanged throughout the experimental germination period.