A comparison of the composition of epicuticular wax from red raspberry (Rubus idaeus L.) and hawthorn (Crataegus monogyna Jacq.) flowers

Epicuticular waxes have been characterised from the flowers of raspberry and hawthorn, on both of which adult raspberry beetles (Byturus tomentosus) can feed. The flower wax from both species had similar alkane profiles and also contained long-chain alcohols, aldehydes and fatty acids. The range of the carbon numbers detected for these classes of compounds was broadly similar in both but the relative amounts of each differed between species. Raspberry flower wax also contained fatty acid methyl esters, a group of compounds that has rarely been detected in plant epicuticular waxes, however, these were not observed in hawthorn flower wax. Long-chain alcohol-fatty acid esters with carbon numbers ranging from C36 to C48 were also detected in both plant species. However, an examination of their constituent acids indicated that in hawthorn the esters based on the C16 fatty acid predominated, whilst in raspberry flower wax, esters based on the C20 fatty acid were most abundant. Both species also contained pentacyclic triterpenoids, which accounted for, on average, over 16 and 48% of the total wax extracted from raspberry and hawthorn flowers respectively. In the former, ursolic and oleanolic acids accounted for over 90% of the pentacyclic triterpenes, whilst hawthorn flower wax, in addition to containing these acids, also contained high relative concentrations of both free and esterified alpha- and beta-amyrins.     

Enhancement of fruity aroma production of Pseudomonas fragi grown on skim milk,whey and whey permeate supplemented with C3-C7 fatty acids

Summary Pseudomonas fragi strain CRDA 037 produced a fruity aroma when grown in skim milk-, whey-and whey permeate-based culture media. The production of the odour-active metabolites was related to the lipid content of these media but was not influenced by the pH of the cultures. Analysis of the fruity aroma revealed that esters of fatty acids were some of the odouractive metabolites. Addition of C₃-C₇ fatty acids to the culture at 0 h stimulated the production of the corresponding fatty acid esters from 12 to 1570 times compared to unsupplemented media. Supplementation of the culture media with the C₃-C₇ fatty acids at 48 h, resulted in a 1.4- to 932-fold increase in the ethyl ester concentration.     

Synthesis and degradation of fatty acid ethyl esters by cultured hepatoma cells exposed to ethanol

Fatty acid ethyl esters are a family of neutral lipids that are the products of esterification of fatty acids with ethanol. Unlike other pathways of ethanol metabolism, ethyl esters are present in numerous human organs which are the targets of ethanol-induced damage. In the present study, we have shown that fatty acid ethyl esters are synthesized by a hepatoma cell line in tissue culture when exposed to ethanol concentrations easily attained by man during social drinking. Unlike alcohol dehydrogenase, the enzyme(s) responsible for synthesis of ethyl esters are membrane-bound and concentrated in the microsomal fraction of rat hepatocytes. In addition, fatty acid ethyl esters are hydrolyzed to free fatty acids and ethanol by membrane-bound enzyme(s) that are enriched in the microsomal and mitochondrial-lysosomal fractions. Intracellular hydrolysis of fatty acid ethyl esters release free fatty acids which are preferentially incorporated into cellular cholesterol esters. Thus, we have shown that a hepatocellular line exposed to concentrations of ethanol easily achieved in man by social drinking utilize endogenous fatty acids to form long-lived ethanol metabolites, fatty acid ethyl esters. Importantly, this family of neutral lipids may act as biochemical mediators of ethanol-induced cell damage, including the changes in cholesterol metabolism noted in chronic alcoholics.     

Oxidative stress and inflammation in renal patients and healthy subjects

The first goal of this study was to measure the oxidative stress (OS) and relate it to lipoprotein variables in 35 renal patients before dialysis (CKD), 37 on hemodialysis (HD) and 63 healthy subjects. The method for OS was based on the ratio of cholesteryl esters (CE) containing C18/C16 fatty acids (R2) measured by gas chromatography (GC) which is a simple, direct, rapid and reliable procedure. The second goal was to investigate and identify a triacylglycerol peak on GC, referred to as TG48 (48 represents the sum of the three fatty acids carbon chain lengths) which was markedly increased in renal patients compared to healthy controls. We measured TG48 in patients and controls. Mass spectrometry (MS) and MS twice in tandem were used to analyze the fatty acid composition of TG48. MS showed that TG48 was abundant in saturated fatty acids (SFAs) that were known for their pro-inflammatory property. TG48 was significantly and inversely correlated with OS. Renal patients were characterized by higher OS and inflammation than healthy subjects. Inflammation correlated strongly with TG, VLDL-cholesterol, apolipoprotein (apo) C-III and apoC-III bound to apoB-containing lipoproteins, but not with either total cholesterol or LDL-cholesterol.In conclusion, we have discovered a new inflammatory factor, TG48. It is characterized with TG rich in saturated fatty acids. Renal patients have increased TG48 than healthy controls.     

One-step extraction of human erythrocyte lipids allowing rapid determination of fatty acid composition

A one-step extraction procedure to directly quantify cholesterol, phospholipids, and fatty acids in red blood cell membranes has been developed. The method uses a single solvent, isopropanol, which extracts lipids and allows the rapid formation of isopropylic esters of fatty acids by acid catalysis. The efficiency of this new technique has been confirmed by comparing yields of cholesterol and total and individual phospholipids with yields obtained following conventional extraction procedures. Moreover, in comparison to the formation of methyl esters, we demonstrate that directly obtained isopropylic esters immediately allow the quantitative determination of fatty acids, without involving the hydrolytic degradation of fatty acids and the oxidation of unsaturated fatty acids.     

FATTY ACID ABNORMALITY IN ADRENOLEUKODYSTROPHY

—Recent clinical and morphological evidence established that adrenoleukodystrophy is a distinct X-linked genetic disorder. Fatty acid compositions of lipids in the brain, adrenal and serum from seven patients were examined. Cholesterol esters of both brain and adrenal contained substantial proportions of fatty acids longer than C₂₂ (11.8–41.9% of total in the brain and 13.4-34.8% of total in the adrenal), while cholesterol esters from normal and pathological control specimens contained very little. These very long chain fatty acids were generally saturated in brain cholesterol esters but significant amounts of unsaturated long chain fatty acids were also present in adrenal cholesterol esters. The long chain fatty acids showed bell-shaped distribution with C₂₅ or C₂₆ at the peak. Ganglio-sides from patients’white matter also showed increased proportions of very long-chain fatty acids, up to 50% of the total. Qualitatively similar but much milder fatty acid abnormalities were also found in galactosylceramide of the brain. On the other hand, fatty acids and fatty aldehydes of brain glycerophospholipids, adrenal free fatty acids, triglycerides and glycerophospholipids were not abnormal. Furthermore, serum cholesterol esters from two patients did not show the long-chain fatty acid abnormality found in brain and adrenal cholesterol esters. Sequential extractions with acetone and hexane established that the characteristic birefringent material in the brain and adrenal is indeed cholesterol esters with very long chain fatty acids. This type of fatty acid abnormality has not been described in other pathological conditions and may well represent the unique biochemical abnormality that is directly related to the fundamental genetic defect underlying adrenoleukodystrophy.     

Effects of various non-esterified fatty acids on the transfer of cholesteryl esters from HDL to LDL induced by the cholesteryl ester transfer protein

The effects of saturated, monounsaturated and polyunsaturated non-esterified fatty acids on the rate of transfer of radiolabeled cholesteryl esters from high density lipoproteins (HDL) to low density lipoproteins (LDL), induced by the cholesteryl ester transfer protein (CETP), have been studied. Human high-density lipoproteins-subfraction 3 (HDL3) containing radiolabeled cholesteryl esters were incubated with LDL at 37 degrees C with or without CETP and in the absence or in the presence of non-esterified fatty acids. Less than 6% of the total radioactivity was recovered in the LDL fraction after incubation of HDL3, and LDL for 3 h at 37 degrees C in the absence of CETP, regardless of whether or not non-esterified fatty acids were added. The addition of CETP to the incubation mixture induced a time-dependent redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. Non-esterified fatty acids were found to alter the rate of transfer of cholesteryl esters induced by CETP. While short chain saturated non-esterified fatty acids (caprylic and capric acids) had no effect on the rate of transfer of cholesteryl esters, the medium and long chain ones (lauric, myristic, palmitic and stearic acids) significantly increased the CETP-mediated transfers from HDL3 to LDL. At low concentrations, unsaturated fatty acids also stimulated the CETP-mediated redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. As the concentration of either oleic, linoleic or arachidonic acids increased to higher levels, a significant proportion of fatty acids remained unassociated with lipoprotein particles. Under these circumstances the transfer process was inhibited. These results show that non-esterified fatty acids can modulate the CETP-mediated transfer of cholesteryl esters from HDL to LDL and that this effect is dependent on both the length and the degree of unsaturation of their monomeric carbon chain.     

Significance of lipoidal estradiol in human mammary tumors

Incubations of p-nitrophenyl fatty acyl esters and estradiol-17 beta fatty acyl 17-esters with porcine esterase, human mammary tumor cytosol and rat uterine cytosol leads to ester hydrolysis of compounds with short chain fatty acids. Esters with long chain fatty acids show no hydrolysis except in the presence of Tween 80. Short chain fatty acid esters have a higher binding potency to the estrogen receptor than long chain fatty acid esters. Extraction of the nuclear receptor peak sedimenting at 4.6S and identification of the steroid showed that about 90% of the radioactivity was associated with estradiol and only 10% with estradiol esters. These studies show that estradiol fatty acyl esters act as a storage form from which estradiol is released by enzymatic hydrolysis.     

Changes of dolichol and dolichyl fatty acyl esters during the germination and development of soybeans.

The amounts of dolichol and dolichyl fatty acyl esters and their composition in various parts of soybean seedlings were determined during germination and development. The dolichol content of cotyledons decreased during germination. Dolichyl fatty acyl esters were identified in cotyledons and the amount was estimated by high performance liquid chromatography. The relative amounts of short-chain dolichols of 15, 16, and 17 isoprene units increased during development of the seedlings. The homologue distribution of free dolichol was different from that of dolichyl fatty acyl esters. The relative amounts of dolichols with 16, 17, and 18 isoprene units were greater in free dolichol than in dolichyl fatty acyl esters. The percentages of long-chain saturated fatty acids in dolichyl fatty acyl esters, specifically 21:0, 22:0, 23:0, 24:0, and 25:0, increased during development. These fatty acids represented more than 40% of the fatty acids in dolichyl fatty acyl esters in stems. These results suggest that dolichyl fatty acyl esters are not a storage form of dolichol. The large accumulation of dolichol and dolichyl fatty acyl esters in the leaves, where photosynthesis takes place, suggests some other function.     

Concentration and fatty acid composition of cholesteryl esters of normal human brain

Abstract— —Cholesteryl esters were isolated from the cerebral cortex and white matter of human brains at different ages, and their concentration and composition determined. The esters were separated from other lipids by chromatography on silicic acid and finally purified by TLC. The fatty acids were converted to the methyl esters by alkaline trans-methylation and analysed by GLC. A TLC method was elaborated for quantitative determination of small amounts of cholesteryl esters in the presence of free cholesterol. The concentration of cholesteryl esters was only 0·1–0·2 per cent of the total cholesterol content of cerebral tissue in older children and adults. During early myelination the concentration was many times greater, especially in the white matter but it never exceeded 2 per cent of the total cholesterol in any subject. The major fatty acids of human brain cholesteryl esters were oleic, palmitic, palmitoleic and arachidonic acid. After completion of myelination, arachidonic acid constituted the major fatty acid. There were fairly small differences in the fatty acid pattern of the cholesteryl esters between grey and white matter, but the concentration of polyunsaturated fatty acids was larger in the grey matter. Cholesteryl esters appear to play an important role in the metabolism of the phosphoglyceride fatty acids in cerebral tissue.     

Quantitative analysis of fatty acid precursors in marine samples: direct conversion of wax ester alcohols and dimethylacetals to FAMEs

To apply fatty acid analyses to the study of foraging ecology and diet determination, all compounds that may be deposited as fatty acids in a predator must be quantified in the prey. These compounds include the usual fatty acids in acyl lipids, but also the alcohols of wax esters and the vinyl ethers of plasmalogens. In routine fatty acid analysis, samples are extracted and transesterified (methylated), resulting in the formation of fatty acid methyl esters (FAMEs); however, fatty alcohols and dimethylacetals (DMAs) are also generated if wax esters or plasmalogens are present. Here, we present a new method using a modified Jones' reagent to oxidize these alcohols and DMAs to free fatty acids (FFAs). These FFAs are then easily methylated and quantitatively recombined with FAMEs from the same sample. This generates a fatty acid signature of prey that is equivalent to that which the predator has available for deposition upon digestion of that prey. This method is validated with alcohol and DMA standards. Its application to typical marine samples is also presented, demonstrating the change in effective fatty acid signature after inclusion of fatty acids derived from wax esters and plasmalogens.     

Fatty acid composition of Simonsiella strains

Gas-liquid chromatography of methyl esters of bound fatty acids extracted from the cells of 48 Simonsiella strains showed that these aerobic, gliding, multicellular-filamentous bacteria have fatty acid profiles of the pattern considered typical of Gramnegative eubacteria. All strains contained predominantly tetradecanoic acid (29.5%), 9-hexadecenoic acid (22.2%), an unidentified acid with an equivalent chain length of approximately 20 carbon atoms (15.8%), and dodecanoic acid (11.4%).Discriminant analysis of the mean relative percentages of 12 fatty acids correctly assigned 94% of the strains to groups based on their source of origin (i.e., the oral cavities of sheep, cat, human or dog); the relative amounts of only 3 of the fatty acids (9-octadecenoic acid, hexadecanoic acid, and tetradecanoic acid) provided most of this discrimination.     

Skin surface lipids of the mole Scalopus aquaticus

Skin surface lipids of the mole Scalopus aquaticus were found to consist principally of squalene (70%), wax esters (15%), and sterol esters (5%), together with small amounts of triglycerides, free fatty acids, free fatty alcohols, and free sterols. Analysis of the fatty acids occurring free and as wax esters and sterol esters showed these to consist of approximately equal amounts of saturated and monounsaturated compounds. The saturated fatty acids consisted predominantly of odd-carbon anteiso and even-carbon straight-chain compounds, with minor amounts of even-carbon iso-branched chains. The unsaturated fatty acids had double bond positions that would have been produced by delta 9-desaturation of C14, C16 and C18 straight chain saturated precursors. Both the free and the esterified fatty alcohols had chain structures corresponding with those of the fatty acids but of somewhat greater average chain length. Discovery of a major proportion of squalene in the sebum of this animal extends the number of non-human species that have this characteristic to four, all of which inhabit a damp environment, suggesting that squalene conveys some biological advantage under these conditions.     

Altered cholesterol ester proportions in embryonic tissues of dystrophic chicken

The concentrations of cholesterol esters in tissues of dystrophic chicken embryos are altered from normal. These changes are accompanied by significant changes in the proportions of the esterified fatty acids (the fatty acid profile). In serum and pectoral muscles there is a shift to a higher proportion of unsaturated fatty acids (in particular 18:1). Thigh muscle esters are little changed and in liver and brain the proportion of unsaturated fatty acids decreases.     

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.     

Fatty acids of astaxanthin esters in krill determined by mild mass spectrometry

Krill is a major source of astaxanthin, which has strong antioxidant activity. Fractions with astaxanthin monoesters and diesters of Antarctic krill Euphausia superba were isolated. Astaxanthin esters were separated by C18-HPLC depending on the number of carbons and double bonds of esterified fatty acid(s). Small amounts of other lipids remained in the samples, but relative molecular masses of carotenoid esters could be measured by field desorption mass spectrometry without fragmentation and interference from contaminant lipids. The fatty acids were determined by calculation of difference between astaxanthin and astaxanthin esters. Only five kinds of fatty acids, dodecanoate, tetradecanoate, hexadecanoate, hexadecenoate and octadecenoate, were detected. Fast atom bombardment mass spectrometry and secondary ion mass spectrometry showed similar spectra. The fatty acid composition in astaxanthin esters was different from those in krill lipids. Therefore, determination of fatty acids in carotenoid esters by a combination of HPLC elution profile and mild mass spectrometry is found to be a useful tool.     

Lipolysis of menhaden oil triacylglycerols and the corresponding fatty acid alkyl esters by pancreatic lipase in vitro: a reexamination

In order to distinguish between possible fatty acid differences during lumenal lipolysis and cellular absorption, we have reinvestigated the in vitro hydrolysis of menhaden oil and its alkyl esters by pancreatic lipase. For this purpose we incubated menhaden oil or its fatty acid methyl and ethyl esters with porcine pancreatic lipase in the presence of bile salts and determined the composition of the released free fatty acids, monoacylglycerols, diacylglycerols, and residual triacylglycerols, or the free fatty acids and residual alkyl esters, respectively, by thin-layer and gas-liquid chromatography. There was significant discrimination against the delta 4- to delta 7-unsaturated fatty acids of both medium and long chain lengths during the hydrolysis of menhaden oil and its fatty acid ethyl esters. In general, the ethyl esters were hydrolyzed 10-50 times more slowly than the corresponding glyceryl esters, depending on the exact ratio of the two substrate types. None of the triacylglycerols or ethyl esters, however, was completely resistant to hydrolysis resulting in an eventual cleavage of all the alkyl esters and presumably all the primary ester bonds in the triacylglycerol molecules. Since the rate of release of the least resistant fatty acid exceeded that of the most resistant acid by only a factor of 6, it is concluded that in the presence of a large excess of lipase the liberated fatty acids would approach the composition of the dietary alkyl or glyceryl esters, as observed during lumenal lipolysis (Yang, L.-Y., A. Kuksis, and J. J. Myher. 1989. Biochem. Cell Biol. 67: 192-204).     

Lumenal hydrolysis of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat

Simple alkyl (ethyl) esters of polyunsaturated fish oil fatty acids have been proposed as dietary supplements, but their relative efficiency of digestion and absorption have not been determined. Using stomach tubes, we gave rats menhaden or rapeseed oils, or the corresponding methyl and ethyl esters, and determined by chromatographic methods the lipid classes and molecular species recovered from the lumen of the jejunum during the first 1 to 2.5 h of digestion. Hydrolysis of menhaden oil resulted in a preferential retention of a high proportion of the polyunsaturated long chain acids in the sn-2-monoacylglycerols and in the residual triacyglycerols, while digestion of rapeseed oil led to a preferential release of free long chain monounsaturated fatty acids. In contrast, hydrolysis of the alkyl (methyl and ethyl) esters of the fatty acids of either menhaden or rapeseed oil resulted in a composition of free fatty acids which was much more representative of the original esters. It was therefore concluded that the differential lumenal liberation of the long chain and polyunsaturated (three or more double bonds) fatty acids from fish and rapeseed oil is largely due to their characteristic distribution between the primary and secondary positions in the glycerol molecule, and to a much lesser extent to a chain length discrimination by pancreatic lipase. This study also shows that the methyl and ethyl esters are hydrolyzed about 4 times more slowly than the corresponding triacylglycerols, which is sufficient to maintain a saturated micellar solution of fatty acids in the intestinal lumen during absorption.     

Biosynthesis of C30 to C56 fatty acids by an extract of Mycobacterium tuberculosis H37Ra.

A 10,800 X g supernatant fraction from disrupted cells of Mycobacterium tuberculosis H37Ra was incubated with [2-14C]malonate to produce labeled long-chain fatty acids upon saponification. These acids were derivatized to the p-bromophenacyl ester and separated into the nonmycolic saturated, monounsaturated, and multiunsaturated esters by argentation thin-layer chromatography. Each of these fractions was then analyzed by high-performance liquid chromatography by using a C18-bonded silica cartridge and a mobile phase of a linear gradient of 0 to 70% p-dioxane in acetonitrile. The results showed that the cell-free system is able to synthesize both saturated and monounsaturated fatty acids of the sizes C30 to C40 and C48 to C56. This latter series was strikingly similar to meromycolic acid, a putative precursor of mycolic acid. When acetate or oleate was used as the labeled substrate, the major products were no longer than C32. When palmitate was used as the labeled substrate, the saturated acids ranged in size from C18 to C32, whereas the monounsaturated products contained C18, C26 to C30 and C40 fatty acids. Fatty acids greater than C40 were also detected. When methyl-labeled S-adenosyl-L-methionine was used as the substrate, the methyl group was incorporated into short-chain and C48 to C56 fatty acids. Unlabeled malonyl-coenzyme A was included in all of these reactions. This cell-free system was not able to synthesize mycolic acid (final product) or its keto derivative (intermediate product). However, since the meromycolate-like C48 to C56 fatty acids were synthesized, we suggest that the present system is able to take the synthesis to a point before the alpha-alkyl condensation reaction.     

Effects of Three Bacterial Infections on Serum Lipids of Rabbits

Alteration of the rabbit serum lipids as a result of three bacterial infections was studied by quantitative thin-layer and gas-liquid chromatography. Anthrax infection slightly changed the serum lipid. Cholesterol did not change, though free fatty acids, triglycerides, and cholesteryl esters doubled, and lecithin increased threefold. Tularemia infection produced drastic changes in the serum lipid content of rabbits, increasing levels of cholesterol over 4-fold, free fatty acids 17-fold, triglycerides 11-fold, cholesteryl esters 2.5-fold, and lecithin almost 3-fold. Pneumococcus infection increased cholesterol 2.5 times, free fatty acids were more than doubled, triglycerides were increased 9.5 times, and lecithin was increased almost 4 times. Gas-liquid chromatographic analysis of the methyl esters of free fatty acids showed only quantitative changes in these acids due to infection. Some possible mechanisms of alteration of serum lipid content are discussed.