Freie und gebundene Sterine in rohen und raffinierten Palmölen,Teil III: Verhalten der sterinhaltigen Lipoproteine bei der Raffination

Free and Bound Sterols in Raw and Refined Palm Oils, Part III: Behaviour of Sterol Containing Lipoproteins during Refining Studies have been carried out about the behaviour of sterol containing lipoproteins during refining. By neutralization and bleaching they are partly separated together with the free fatty acids. One part, however, is splitted mainly during deodorization. Thus content and composition of lipoproteins are changed during refining. The sterols released from lipoproteins are determined together with free sterols. Because of their high cholesterol content increased cholesterol values can be found in sterol determination of refined palm oils.     

Palmöle und Palmöl-Fraktionen und ihre analytische Differenzierung

Palm Oils and Palm Oil Fractions and their Analytical Differentiation Due to the increasing production of palm oils and the correlating production of palm oil fractions there is a need to differentiate analytically between palm oils and palm oil fractions. The analytical differentiation of these products on the one hand and the detection of unwanted additions and mixtures on the other hand is intended to be achieved. Palm oils of different origin and palm oil fractions, fractionated by different procedures, were examined in this context. The composition of total fatty acids, fatty acids in 2-position of the triglycerides, slip point, iodine value, composition of triglycerides by gaschromatography as well as saturated triglycerides were determined. The slip point, which can easily be determined, and the palmitic acid content in 2-position of t he triglycerides proved to be most suitable for the analytical practice.     

Seasonal Variations of Lipid Content and Composition in <Emphasis Type="Italic">Perna viridis</Emphasis>

The total lipid content, composition of main lipid classes, composition of sterols and composition of fatty acids in the main glycerolipids of Perna viridis were analyzed through four seasons using TLC-FID and GLC. Mussel samples were collected during different seasons between 2003 and 2004 from Shengsi Island, Zhejiang Province, China and stored frozen prior to freeze-drying and lipid extraction. Ten grams of dried mussel powder of each season were analyzed. Total lipid content ranged from 14.5 g/100 g in spring month to 7.8 g/100 g dried mussel powder in autumn month. The predominant lipid in spring month was triacylglycerol (TAG), however, in the other three seasons the phospholipids (PL) was the main lipid class. The most abundant fatty acid in TAG, PL and phosphatidylcholine (PC) was 16:0, with the summer samples having the highest proportion (24-30% of total fatty acid) and winter the lowest (14-22%). In phosphatidylethanolamine (PE), the spring samples had the highest proportions of 16:0. The predominant polyunsaturated fatty acids (PUFA) were 22:6n-3 and 20:5n-3 in TAG, PL, PE and PC (25-40%). The proportions of 22:6n-3 and 20:5n-3 were higher in spring than in other seasons in PL and PE. There were nine sterols identified, with cholesterol being the predominant sterol, and other main ones were desmostersol/brassicasterol and 24-methylenecholesterol. Proportions of other fatty acids in different lipid fractions and the sterol compositions as well also varied seasonally. There were subject to the seasonal variations. Differences in lipid content and composition, fatty acid composition in different lipid fractions may be caused by multiple factors such as lifecycle, sex, variation of plankton in different seasons and temperature, which could influence physiological activities and metabolism.     

Freie und gebundene Sterine in rohen und raffinierten Palmölen,Teil I: Gehalt und Zusammensetzung der freien Sterine,Sterinester, freien und acylierten Steringlucoside

Free and Bound Sterols in Raw and Refined Palm Oil, Part I: Content and Composition of Sterols, Sterol Esters, Free and Acylated Sterol Glucosides A method for separation and quantitative determination of free sterols, sterol esters, free and acylated sterol glucosides in fats was developed and applied to the investigation of raw and refined palm oil. During refining the content and the composition of all 4 sterol fractions are varying characteristically. Some samples showed an evident increase of the cholesterol level during certain refining steps. Since none of the 4 investigated sterol fractions has such a high cholesterol level, sterols must be present in bound form, which can be liberated during refining and can be determined afterwards by conventional test methods.     

Freie und gebundene Sterine in Pflanzenfetten

Free and Bound Sterols in Vegetable Fats Investigations of 15 different vegetable fats have shown that lipoproteins, detected in raw palm oil, occur in other fats, too. The cholesterol content in the sterols of lipoproteins is not always the same; but it is in any case higher than in other sterol containing fractions of these fats. The investigation of the free sterols, sterol esters, sterol glycosides and sterol containing lipoproteins in these 15 fats shows that cholesterol occurs more or less in all vegetable fats, but in an analytically difficult accessible way.     

Detection of adulteration

A program of work is in progress to establish the levels and ranges of fatty acids and other components present in the major edible vegetable oils. Authentic samples from the major producing areas for such oil have been obtained and analyzed. In the case of palm oil, ranges of the fatty acid composition and of the acids at the triglyceride 2-position, have been obtained for about 40 samples. These data were used to calculate enrichment factors, and triglyceride carbon number compositions, using a small computer program. Comparison with experimentally determined carbon number compositions were then made. Good correlations were found for whole unadulterated oils, but not for oil fractions. Unfortunately, these differences were insufficient to detect contamination of palm oil by 10 or 20% levels of other oils, or of palm fractions. Compositional ranges of sterols and tocopherols have also been determined on a selection from the original set of palm samples. Work on sunflower seed and groundnut oils has followed the same lines, particular attention having been paid to linolenic acid and, in the case of groundnut oil, also erucic acid, levels. Some groundnut kernels were found to have an oil with a component which cochromatographed with methyl erucate during fatty acid determination. This unknown constituent was studied by gas chromatography-mass spectrometry, and is thought to comprise a mixture of epoxy fatty acids. Analysis of the triglyceride fraction isolated from groundnut oil by thin layer chromatography removes this unknown constituent, and simplifies interpretation of the fatty acid composition of groundnut oil.     

Chromatographic characterization of internal polar lipids from wool

Wool internal polar lipids were isolated and separated into different fractions based on polarity. Qualitative and quantitative analyses of the different fractions were performed by thin-layer chromatography and thin-layer chromatography coupled to flame-ionization detection, respectively. Cholesterol esters, free fatty acids, sterols, ceramides, glycosylceramides, and cholesterol sulfate were the main components, with ceramides being in the highest proportion. The fatty acid composition of ceramides and glycosylceramides was determined by gas chromatography/mass spectrometry. As for other keratinized tissues, long-chain fatty acids predominated in comparison to either free fatty acids or phospholipid-linked fatty acids; in both cases, stearic and lignoceric acids were the most abundant fatty acids, and a low amount of 18-methyleicosanoic acid was found. This work opens new avenues in the study of lipid rearrangement in more complex and realistic vesicle structures than conventional liposomes.     

Fatty acid composition of individual plasma steryl esters in phytosterolemia and xanthomatosis

The bulk of the plasma plant sterol in phytosterolemia occurs in the esterified form and is carried mostly in the low and high density lipoproteins. We have determined the fatty acid composition of the individual plasma steryl esters from a newly discovered subject with phytosterolemia and xanthomatosis. For this purpose the intact steryl esters were subject to high temperature gas liquid chromatography (GLC) on a polar capillary column, which separated the major esters on the basis of molecular weight and degree of unsaturation of the fatty acids. The saturated and unsaturated sterols esterified to saturated, monoenoic, dienoic and tetraenoic fatty acids were identified by GLC analysis of the sterol moieties of the corresponding AgNO₃-TLC fractions of the steryl esters. The GLC results were confirmed by reversed phase high performance liquid chromatography combined with mass spectrometry via direct liquid inlet interface. It was found that, in general, each fatty acid was esterified to the same complement of sterols, and that the esterified sterols possessed a composition comparable to that of the free plasma sterols, which was comprised of about 75% cholesterol, 6% campesterol, 4% 22,23-dihydrobrassicasterol and 15% β-sitosterol. The fatty acid composition of the steryl esters differed from that of the 2-position of the plasma phosphatidylcholines, which contained significantly less palmitic and oleic and more linoleic acid. On the basis of these results and a review of the literature it is suggested that the plasma cholesteryl and plant steryl esters in phytosterolemia originate from both synthesis in plasma via the lecithin-cholesterol acyltransferase and synthesis in tissues via the acylCoA-cholesterol acyltransferase.     

Determination of the adulteration of butter

The adulteration of butter is a serious problem due to economic advantages taken by replacing expensive milk fat with cheaper oil without informing the customers. The authentication of milk fat methods include analysis of bulk components, especially triacylglycerols, fatty acids, sterols and tocopherols. Fatty acid and sterol composition was analysed by using GC‐MS. TAG and tocopherol profiles were examined by HPLC with diode array (DAD) and fluorescence detectors (FLDs). In addition, identification of selected TAG of butter fat was conducted by LC‐atmospheric pressure chemical ionisation (APCI)/MS technique. The lipid composition of 16 different butters available on Polish market were investigated. The cholesterol content in butter fat ranged from 176.8 to 264.8 mg/100 g of fat and in two samples of milk fat β‐sitosterol was found. The total saturated fatty acid (SFA) content in milk fat was 67.1–73.5%, monounsaturated fatty acid 24.5–30.5% and polyunsaturated fatty acid was 1.2–2.0%. Abnormalities in fatty acid profiles, e.g. high concentration of linoleic fatty acid, were found in two butters. These abnormalities were also determined in TAG profiles. The examination of tocopherols in butter fat confirmed that two products were adulterated by the addition of plant oils because they contained δ‐tocopherol which is typical for plant origin foodstuffs. The methods described are useful for investigating milk fat adulterations, and the most efficient are analysis of sterols and tocopherols composition. Practical applications: The described methods are useful for investigating adulteration of milk fat. Traditional strategies rely on examination of fatty acids methyl esters and TAG; these methods have some disadvantages. Due to the variability of fatty acid composition of milk fat and because TAG analysis is complex and time consuming, FA analysis is not an efficient approach for butter authentication. The most efficient method for butter authentication is qualitative and quantitative analysis of sterols and tocopherols. This analysis will determine if components of plant origin were used for butter production.     

Comparison of total fat,fatty acids,cholesterol, and other sterols in mayonnaise and imitation mayonnaise

Nine brands of mayonnaise and five brands of imitation mayonnaise were purchased from supermarkets in the Washington, DC, area. The samples were analyzed for total fat, fatty acids, sterols, and moisture. Little variation in total fat and saturated fatty acid values was observed among the brands of mayonnaise. The polyunsaturated fatty acid content of mayonnaise ranged from 28.0 to 47.9 g/100 g product. The cholesterol levels were divided between two ranges, 50-55 and 75-79 mg/100 g product. In contrast, there was wide variation in the lipid composition of the different brands of imitation mayonnaise. The total fat values for these products varied from 14.3 to 50.4 g/100 g product. The cholesterol content varied between 0 and 72 mg/100 g product; the latter figure equals the cholesterol content of many of the mayonnaise samples.     

Chemoattraction ofBiomphalaria glabrata (Gastropoda: Planorbidae) to lipid standards and lipophilic factors in leaf lettuce and Tetramin

Chemoattraction of individualBiomphalaria glabrata snails for lipid standards and lipophilic fractions of leaf lettuce and Tetramin were studied in a Petri dish bioassay. Snails were more significantly attracted to a whole Tetramin lipophilic fraction than that of leaf lettuce. Thin-layer chromatography showed that major neutral lipid fractions in Tetramin were triacylglycerols, free fatty acids, and free sterols, and in leaf lettuce were free fatty acids and a mixed free sterol-chlorophyll fraction. Snails were significantly attracted to both the free fatty acid and free sterol fractions from Tetramin, but only to the free fatty acid fraction from leaf lettuce. Snails were significantly attracted to a mixed lipid standard containing equal amounts of phosphatidylcholine, cholesterol, oleic acid, triolein, and cholesteryl oleate. Of four individual neutral lipid standards tested, i.e., cholesterol, oleic acid, triolein, and cholesteryl oleate, snails were only attracted to cholesteryl oleate.     

The Sterols and Fatty Acids of Delphinium denudatum Roots

The content and composition of sterols and fatty acids of Delphinium denudatum roots, which are used in the Indian Unani system of medicine, were determined. The sterols were composed almost entirely of campesterol, stigmasterol and sitosterol. Trace amounts of cholesterol and Δ⁵-avenasterol were also detected. Characteristic higher plant fatty acids were also present.     

Lipid composition of further purified bovine liver nuclear membranes

The lipid content, distribution and fatty acid composition of highly purified bovine liver nuclear membranes was determined and compared to those of microsomes prepared in parallel. Contrasted with microsomes, nuclear membranes while containing nearly the same levels of lipid had more cholesterol and total neutral lipid and less phospholipid. Phospholipid and neutral lipid patterns generally were similar for the two types of membranes. The same fatty acids, in similar proportions, were observed in respective total lipid, total polar lipid, phosphatidyl choline and phosphatidyl ethanolamine fractions of the two membrane types. The microsomal lipid fractions contained slightly greater percentages of unsaturated fatty acids. With respect to previous results from preparations contaminated with nonmenbranous nuclear material, purified fractions contained more total lipid on a protein basis and more total unsaturated fatty acids. Only minor differences in levels and distribution of phospholipids and neutral lipids were observed between the crude and highly purified fractions. Purdue University AES Journal Paper No. 4482.     

The distribution of lipids and sterols in cell types from the marine spongePseudaxinyssa sp

The sponge Pseudaxinyssa sp., unique in sterol and fatty acid composition, was cellularly dissected into fractions enriched in each of the major cell types present in the sponge: microbial symbionts (cyanobacteria), small sponge cells (pinacocytes and choanocytes), and large sponge cells (archeocytes and cyanophytes). Three phototrophic microbial symbionts were also isolated from the cell fractions and grown in culture. An unsymmetrical distribution of fatty acids and sterols was observed for the sponge cells: small cells contained larger quantities of long chain fatty acids (greater than C24) and smaller quantities of sterols than were present in the larger sponge cells. Moreover, the rare sterols 24-isopropylcholesterol predominated in the smaller sponge cells, whereas its 22-dehydro analog predominated in the larger sponge cells. Long chain fatty acids and sterols were not detected in the cultured microbial symbionts. This constitutes the first report of lipid variability according to cell type for this most primitive group of Metazoa.     

<Emphasis Type="Italic">Oenocarpus bataua</Emphasis> Mart. (Arecaceae): Rediscovering a Source of High Oleic Vegetable Oil from Amazonia

The fatty acid (FA) composition of Oenocarpus bataua oil from 38 samples collected over a large geographical range (i.e. French Guiana and Peru) was analyzed. Fifteen fatty acids were obtained from the mesocarp of this palm species. Oleic (72.7%) and palmitic (18.1%) acids were the predominant FAs. Minor FAs were cis-vaccenic acid (2.3%), linoleic acid (1.9%), stearic acid (1.7%), palmitoleic (0.9%) and alpha-linolenic acid (0.8%). The mean lipid content of the dry mesocarp was 51.6%. The O. bataua oil samples analyzed were remarkably rich in α-tocopherol. By contrast, the other fractions of the unsaponifiable matter (sterols, carotenoids) did not show any noteworthy specificity in comparison with common vegetable oils. However, the particularly high percentage in Δ5-avenasterol of O. bataua oil could serve as a marker for its authentication. Results are discussed in terms of the potential nutritional value of O. bataua oil.     

Sterol and fatty acid composition of neutral lipids ofParatenuisentis ambiguus and its host eel

The sterol composition of free sterol and steryl ester fractions of the fish parasiteParatenuisentis ambiguus was determined. In addition, the fatty acid composition of various neutral lipid classes, i.e., wax esters, steryl esters, triacylglycerols and free fatty acids, as well as the composition of the 1-O-alkyl moieties of total ether glycerolipids of the parasite, were investigated. The results of these studies were compared with those obtained on the intestinal tract tissue of its host, the eel (Anguilla anguilla). Cholesterol is the major sterol in bothP. ambiguus andA. anguilla. However, the sterols ofP. ambiguus contain high proportions (>20%) of other sterols, such as campesterol and various dehydrosterols. [e.g., 7-dehydrocholesterol and cholesta-5,22(E)-dienol]. The presence of these minor sterols agrees with the known biotransformations of exogenous sterols in various helminths. Considerable differences are found in the fatty acid composition of neutral lipid fractions, as well as the total lipid extract from the endoparasite as compared to the host tissue. In particular, eicosapentaenoic acid (20∶5n−3), other polyunsaturated fatty acids, such as 20∶4n−6, 22∶5n−3 and 22∶6n−3, as well as long-chain saturated fatty acids, such as 20∶0, are generally enriched in the neutral lipid fractions of the parasite as compared to those of infected eel intestine. The analysis of ether glycerolipids revealed that 1-O-hexadecyl (16∶0) and 1-O-hexadecenyl (16∶1) moieties were present in similar proportions in the ether lipids of bothP. ambiguus and eel intestine, whereas 1-O-octadecyl (18∶0) moieties are more prominent in the parasite and 1-O-octadecenyl (18∶1) moieties in the eel. The results of these studies show thatP. ambiguus has specific mechanisms for the regulation of the sterol and fatty acid composition of its neutral lipids. Dedicated to Professor Helmut K. Mangold on the occasion of his 70th birthday.     

Lipid composition and peroxide levels of mucosal cells in the rat large intestine in relation to dietary fat

To examine whether dietary fat alters membrane lipid composition and peroxidation of polyunsaturated fatty acids in “non-proliferative” and “proliferative” cells in the large intestine, Sprague-Dawley rats were fed diets providing a polyunsaturated-to-saturated fatty acid ratio of 1.2 or 0.3 at a high or low level of fat intake for a 25-day period. Cell populations were isolated and the effect of dietary fat on membrane polyunsaturated fatty acid content and peroxide levels was determined. Neither fat level nor fatty acid composition of diet influenced total cholesterol, total phospholipids, and percentage of phospholipid classes in membrane phospholipids. Feeding the high fat and/or high polyunsaturated-to-saturated fatty acid ratio diet increased polyunsaturated fatty acid content of mucosal cell phospholipids. Increase in polyunsaturated fatty acid content was paralleled by a decrease in the monounsaturated fatty acid content of mucosal cell phospholipids. Membrane content of total saturated fatty acids was not significantly affected by diet. Variation in phospholipid fatty acid composition between “non-proliferative” and ”proliferative” cells was observed. Lipid peroxide levels in mucosal cell lipid fractions were altered by dietary fat treatment. Animals fed high fat diets, compared to groups fed low fat diets, exhibited higher membrane peroxide levels when results are expressed as nmol/mg protein. Higher peroxide levels were observed in mucosal cells for rats fed high polyunsaturated-to-saturated fatty acid ratio diets when results were expressed per nmol of phospholipid. It is concluded that changes in fat level and fatty acid composition of the diet alters the mucosal cell membrane lipid composition in the rat large intestine and influences susceptibility of mucosal cell lipid to peroxidation. Further research is required to delineate which dietary factors—fat level, polyunsaturated-to-saturated fatty acid ratio, or both—have a primary influence on the degree of lipid peroxidation.     

Studies in palm oil crystallization

The melting and crystallization behaviors of palm oil were examined by nuclear magnetic resonance, differential scanning calorimetry, and plasticity measurements and were correlated with composition and chemical characteristics. Fractionation into high melting and low melting components is adversely affected by increase in free fatty acids (FFA), diglyceride content, and degree of oxidation, and hence, for an oil that is to be fractionated, both hydrolysis and oxidation should be kept to a minimum. Palm oil with very low FFA content, obtained from ripe, unbruised fruit, contains substantially more diglycerides than would be expected from the level of FFA present. Liquid and solid palm oil fractions manufactured in the producing countries showed large variation in characteristics, due in part to the use of various fractionation processes. The characteristics used to define the quality of palm oil are also applicable to its fractions.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

Determination of sterols, oxysterols, and fatty acids of phospholipids in cells and lipoproteins: A one-sample method

In addition to fatty acids, especially polyunsaturated species, cholesterol oxidizes and leads to various oxygenated derivatives, named oxysterols. They display a wide range of adverse biological properties. Monitoring oxysterols is important in the evaluation of the potential risks associated with lipid oxidation. In the present study, a quick and reliable method was developed for analysis of oxysterols, sterols, and fatty acid composition of phospholipids in the same biological sample. Total lipid extraction was determined after addition of several internal standards (epicoprostanol for sterols, 19-hydroxy-cholesterol for oxysterol and di-heptadecanoyl-phosphatidylcholine for phospholipid fatty acids). Cold acetone-mediated precipitation was then used to fractionate sterols from phospholipids. The phospholipid-containing precipitate was transmethylated for fatty acid analysis by gas chromatography. The sterol- and oxysterol-containing phase was saponified under mild conditions to avoid artificial oxysterol generation and was analyzed by gas chromatography after derivatization into trimethylsilyl ethers. The overall procedure was found to be specific with good recovery and reproducibility for sterols, oxysterols [mean coefficient of variation in percent (CV), 11.3%] as well as phospholipid fatty acids (CV, 5.6%). This procedure has been used to document in vitro free radical treated-human low-density lipoproteins and erythrocytes. Results demonstrated that this method is a useful tool in assessing qualitative and quantitative differences in oxysterols and phospholipid fatty acid patterns attributed to lipid oxidation.