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Plant cells in culture are capable of incorporating exogenous 1-O-alkyl-sn-glycerols into various neutral and ionic ether lipids. 1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholines, the major class of compounds thus formed, are used for the preparation of platelet activating factor (PAF) in high yields. Similarly, the prochiral 2-O-alkyl-sn-glycerols are transformed to chiral 2-O-alkyl glycerophospholipids from which compounds can be obtained that exhibit antiviral activity in plant and animal cells. Reaction of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines with phospholipase D in the presence of ethanolamine leads to 1-O-alkyl-2-acyl-sn-glycero-3-phosphoethanolamines, which serve as starting material for the preparation of 1-O-alkyl-2-acyl-sn-glycero-3-phospho-(N-acyl)ethanolamines, compounds known to have antitumor activity.

Biotransformation of rac-1(3)-O-hexadecylglycerol by photomixotrophic rape (Brassica napus) cells in suspension culture leads to 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines and small proportions of other ether lipids, e.g. 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamines. Reaction of the hexadecylacyl-glycerophosphocholines with ethanolamine in the presence of phospholipase D from Streptomyces chromofuscus yields additional hexadecylacylglycerophosphoethanolamines. Partial hydrolysis of the combined hexadecylacylglycerophosphoethanolamines followed by reacylation of the resulting lyso compound with palmitic anhydride gives 1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phospho-(N-palmitoyl) ethanolamine, a nontoxic ether glycerophospholipid with antitumor activity. The corresponding 1-O-tetradecyl,1-O-octadecyl, and 1-O-[(Z)-9'-octadecnyl] derivatives are prepared similarly.

Radioactivity from cis-9-[1-14C]octadecenol, injected intravenously into rats bearing neurosarcoma, is incorporated to a significantly greater extent into tumor than into muscle. In the lipids of both tissues, radioactivity is incorporated predominantly into the acyl moieties, rather than into the alkyl or alk-1-enyl moieties, of diradylglycerophosphocholines, diradylglycerophosphoethanolamines, and triradylglycerols.

An equimolar mixture of homologous saturated alcohols (13:0, 15:0, 17:0, 19:0) and an equimolar mixture of vinylogous alcohols (19:0, 19:1, 19:2) were fed to two groups of rats. All of the odd-numbered alcohols were incorporated into the ionic alkoxylipids of the rats' small intestine. Pronounced quantitative differences in the distribution of the various odd-numbered alkyl and alk-1-enyl moieties of choline phosphoglycerides and ethanolamine phosphoglycerides gave evidence to differences in the specificity of the enzyme system catalyzing the biosynthesis of alkoxylipids from long-chain alcohols.

Heterotrophic cell suspension cultures of soya (Glycine max) and photomixotrophic cell suspension cultures of rape (Brassica napus) were incubated with cis-9-[1-(14)C]octadecenol for 3-48 h. It was found that under aerobic conditions large proportions of the alcohol are oxidized to oleic acid, which is incorporated predominantly into phospholipids, whereas up to 30% of the substrate is esterified to wax esters. This is true for both the heterotrophic and the photomixotrophic cell suspension cultures, but the metabolic rates are much higher in the latter. Under anaerobic conditions only small proportions of the radioactively labeled alcohol are oxidized to oleic acid, whereas a major portion of the alcohol is esterified to wax esters both in heterotrophic and photomixotrophic cultures. Incubations of homogenates of photomixotrophic rape cells with labeled cis-9-octadecenol showed that pH 6 is optimum for the formation of wax esters. This monounsaturated alcohol is preferred as a substrate over saturated longchain alcohols, whereas short-chain alcohols, cholesterol, and glycerol are not acylated. Incubations of an enzyme concentrate from a homogenate of rape cells with unlabeled cis-9-octadecenol and [1-(14)C]oleic acid, or [1-(14)C]stearoyl-CoA, or di[1-(14)C]palmitoyl-sn-glycero-3-phosphocholine showed that acylation of the longchain alcohol proceeds predominantly through acyl-CoA. Direct esterification of the alcohol with fatty acid as well as acyl transfer from diacylglycerophosphocholine could be demonstrated to occur to a much smaller extent.

Techniques used in the quantitative evaluation of thin-layer chromatograms are compared. The quantitation of tubular thin-layer chromatograms by means of vapor-phase detectors is emphasized, and typical examples of the use of this method are outlined. Applications of tubular thin-layer chromatography (TTLC) in the quantitative analysis of fish liver oils are described.

Some naturally occurring as well as synthetic ether lipids are biologically active. In certain cases, the effects of these substances are enhanced, in others, they are inhibited by compounds that were isolated from natural sources or prepared by chemical synthesis. The biotransformation of natural or "unnatural" ether lipids in microorganisms, plant or animal tissue also can lead to substances that elicit biological effects. The production of such compounds through various biotechnological techniques is a field wide open for future exploration. In addition to animal cell cultures, plant cell cultures may become useful tools in biomedical studies concerned with ether lipids.

Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-[1'-14C]hexadecyl-sn-glycerol or rac-1-O-[1'-14C]hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-[1'-14C]hexadecyl-sn-glycero-3-phosphocholine. 1-O-[1'-14C]Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity.

The distribution of radioactivity from intravenously administered cis-9[1-14C]octadecenol into various tissues of the rat was studied as a function of time. The pattern of incorporation of radioactivity into alkyl, alk-1-enyl and acyl moieties of the lipids in heart, lungs, liver, intestine, kidney, brain and plasma revealed that oxidation of the long-chain alcohol and esterification of the resulting fatty acid to a wide variety of lipids are by far the most predominant reactions. Acylation of the long-chain alcohol is observed especially in liver, which appears to be the major site of biosynthesis of wax esters. Alkylation of the long-chain alcohol to alkoxylipids occurs in most tissues, most predominantly in the heart.

The environmental conditions prevailing during growth of plant tissue cultures affect the concentration of certain lipid classes and the fatty acid patterns of the total lipids. Irrespective of whether the cultures are grown under continuous illumination or in the dark, aerated cultures contain larger proportions of sterols, steryl esters, steryl glycosides and various sterylglycolipids and lower concentrations of squalene than non-aerated cultures. The lipids of the latter contain larger proportions of saturated very long-chain fatty acids than those of the former cultures.

1-O-Alkyl-2,3-diacyl-sn-glycerols, the major constituents of ratfish (Chimaera monstrosa) liver oil, serve as starting material for the preparation of 1-O-alkyl-sn-glycero-3-phosphocholines, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines, 1-O-alkyl-2-O-methyl-sn-glycero-3-phosphocholines, and 1-O-alkyl-2-O-methyl-sn-glycero-3-beta-D-glucopyranosides. Catalytic tritiation of the unsaturated alkyl moieties in these biologically active ether lipids affords the corresponding 3H-labeled substances.

Defatted meals of two new varieties of rapeseed, Brassica napus, Erglu and Lesira, and protein isolates prepared therefrom, were fed to chicks that had been depleted of their embryonic protein reserve. First, the animals were fed at a 12% protein level, half of it from rapeseed meal, or a rapeseed protein isolate and, subsequently, at a 20% level, all of it from rapeseed products. Feed consumption, weight gain and protein efficiency ratio revealed better performance of the protein isolates as compared to the corresponding meals. This is attributed to the favorable amino acid patterns of these isolates and the absence of glucosinolates. Erglu meal, too, is eminently suitable as a supplement to chicken feed, whereas Lesira meal, although its amino acid pattern is adequate and similar to that of Erglu meal, showed detrimental effects, probably due to its high glucosinolate content.

Heterotrophically grown cell suspension cultures of soya (Glycine max L.) were incubated with two different mixed substrates consisting of positional isomers of either cis-[1-(14)C]octadecenoic acids (Δ8 to δ15) or trans-[1-(14)C]octadecenoic acids (Δ8 to Δ16), each with known composition. With both substrates, about one-fourth of the radioactivity supplied was incorporated into the diacylglycerophosphocholines, while another one-fourth of the radioactivity was almost equally distributed between diacylglycerophos-phoethanolamines and triacylglycerols. All the positional isomers of cis-and trans-octadecenoic acids supplied to the cells were readily incorporated into various classes of glycerolipids. None of the octadecenoic acids was isomerized, elongated or desaturated during incubation. From the cis-octadecenoic acids, only the naturally occurring Δ9-isomer (oleic acid) was preferentially incorporated into position 2 of diacylglycerophosphocholines, diacylglycerophospho-ethanolamines, and triacyglycerols; all the other isomers exhibited a strong affinity for position 1 of the glycerophospholipids and positions 1 and 3 of the triacylglycerols. From the trans-octadecenoic acids, only the Δ9-isomer (elaidic acid) was preferentially incorporated into position 2 of diacylglycerophospho-cholines and triacylglycerols; all the other isomers preferred position 1 and positions 1 and 3, respectively, of these lipids. In diacylglycerophospho-ethanolamines, however, each of the trans-octadecenoic acids, including the Δ9-isomer, exhibited a strong affinity for position 1. Apparently, the enzymes involved in the incorporation of exogenous monounsaturated fatty acids into membrane lipids of plant cells can recognize the preferred substrate in a mixture of closely related isomers.

Unsaturated platelet-activating factor (PAF) aggregates thrombocytes of healthy female volunteers and releases within 1 min up to 30.95% of the platelet serotonin. Indomethacin does not inhibit the aggregation but reduces the release of serotonin induced by unsaturated PAF in citrated platelet-rich plasma (PRP). Creatinephosphate combined with creatinephosphokinase (CP/CPK) inhibits the second phase, whereas xylocain inhibits the first and second phase of aggregation induced by unsaturated PAF. Hirudine shows no influence on the aggregation induced by unsaturated PAF.

Three different tests on houseflies (Musca domestica L.) revealed that both pheromone-free pseudoflies and male partner flies exhibit in the presence of mixtures of long-chain (Z)-9-alkenes or pure (Z)-9-tricosene enhanced releasing effects for two optical cues, which stimulate male houseflies to mating strikes and houseflies of both sexes to aggregation.

Defatted meals were prepared from two new varieties of rapeseed, the 'high-glucosinolate' variety, Brassica napus, Lesira, and the 'low-glucosinolate' variety, Brassica napus, Erglu, by each of the following processes: (1) defatting of the ground seed with hexane; (2) extraction of the myrosinase-deactivated ground seed with 70% aqueous acetone for the removal of glucosinolates followed by defatting with pure acetone, and (3) autolysis of the ground seed for the decomposition of glucosinolates followed by defatting and simultaneous removal of the decomposition products with hexane. The defatted meals obtained after extraction with aqueous acetone of autolysis contain very little glucosinolates and their degradation products as compared to those obtained by defatting with hexane only. The contents of available lysine and of phytates in the meals are, to some extent, affected by the treatment for the removal of glucosinolates. The rapeseed meals were fed at different levels to protein-depleted chicks and weanling rats. The animals fed low-glucosinolate meals prepared both from high-glucosinolate and low-glucosinolate varieties of rapeseed gave superior performance with regard to the feed conversion and protein efficiency ratio as compared to the animals fed the corresponding meals which were obtained simply by defatting the seeds with hexane. The levels of thyroxine (T4) and triiodothyronine (T3) in the serum of rats fed low-glucosinolate meals indicated normal function of the thyroid, whereas those in rats fed high-glucosinolate meals revealed hypothyroidism of the animals.

Lipids containing hydroxy fatty acids or hydroxyacyl moieties are acetylated with [1-14C]acetic anhydride or [3H]acetic anhydride, and the content of hydroxy fatty acids or hydroxyacyl moieties is estimated from the specific radioactivity of the acetylated products with respect to that of radioactive standards, such as radioacetylated ricinoleic acid or triricinoleoylglycerol. Mixtures of triacylglycerols containing one, two and three hydroxyl groups per molecule are derivatized in a similar manner, and the resulting acetates are fractionated by thin layer chromatography according to the number of acetate groups per molecule. The relative proportion of each type of triacylglycerols in the mixture is estimated from the distribution of radioactivity in the various fractions. Applications of these techniques are demonstrated by the analysis of several seed lipids.

Photomixotrophic cell suspension cultures of rape, Brassica napus, were incubated with rac-1-O-[1'-(14)C]hexadecylglycerol. Radioactivity was incorporated predominantly into choline glycerophospholipids. Prolonged incubation led also to considerable proportions of labeled ethanolamine glycerophospholipids. In addition to these ionic lipids,isomeric hexadecylacylglycerols as well as hexadecyldiacylglycerols were formed. About a third of the hexadecylglycerol supplied as substrate was cleaved within 48 h incubation. The palmitic acid formed by oxidative cleavage of the substrate was incorporated predominantly into choline glycerophospholipids, ethanolamine glycerophospholipids, and triacylglycerols. Incubation of an equimolar mixture of homologous saturated rac-1-O-[1'(14)C]alkylglycerols (C14, C16, C18, C20) with rape cells showed that alkylglycerols with alkyl moieties having 16 and 18 carbon atoms were incorporated preferentially. Incubation of labeled hexadecyglycerol with a homogenate of rape cells led also predominantly to choline glycerophospholipids; highest yields were obtained at pH 7. Neither the 1-O-alkyl moieties in choline glycerophospholipis nor those in ethanolamine glycerophospholipids were desaturated to 1-O-alk-1'-enylmoieties. The results of these experiments led to the following conclusions: (1) The acylation of 1-O-alkylglycerols to isomeric alkylacylglycerols is catalyzed by two acyltransferases differing in their specificity with regard to the chain length of the alkyl moiety in the substrate. (2) CDP-Choline: diacylglycerol cholinephosphotransferase and CDP-ethanolamine: diacylglycerol ethanolaminephosphotransferase are two enzymes differing in various respects. Cholinephosphotransferase exhibits a much higher affinity for 1-O-alkyl-2-O-acylglycerols than ethanolaminephosphotransferase. The two enzymes show marked differences with regard to their specificity for 1-O-alkyl-2-O-acylglycerols differing in the chain lengths of their alkyl moieties.

The lipids of Chlorella fusca are composed of the ester lipids typical of photosynthetically active cells. In addition, there occurs a class of less common ether lipids, the biologically active 1(3),2-diacylglycero-3(1)-O-4'-(N,N,N- trimethyl)homoserines, at a level of about 1.3% of total lipids. The acyl moieties of the total lipids include saturated as well as mono-, di- and tri-unsaturated species with chain lengths of 16 and 18 carbon atoms, the major constituents being palmitic and oleic acids. In both the diacylglycerophosphocholines, i.e., the major class of ester phospholipids, and the diacylglycero-4'-O-(N,N,N-trimethyl)homoserines palmitic acid is located predominantly at position 1 of the glycerol backbone, whereas oleic acid is almost equally distributed between positions 1 and 2; palmitoleic and polyunsaturated fatty acids are esterified preferentially at position 2. Incubation of C. fusca cultures with 14C-labeled fatty acids leads to their rapid incorporation into various lipid classes. Oleic and palmitic acids are incorporated at a faster rate than stearic acid (18:1 greater than 16:0 much greater than 18:0). 1,2- and 1,3-Diacylglycerols are the most prominent intermediates of early metabolism of the exogenous fatty acids. In the course of time, a steady decrease of radioactive 1,2-diacylglycerols is observed that is accompanied by an increase in labeled triacylglycerols, diacylglycerophosphocholines, and diacylglycero-O-(N,N,N-trimethyl)homoserines. The stereospecific distribution of acyl moieties in the diacylglycero-O-(N,N,N-trimethyl)homoserines in C. fusca indicates that these ether lipids are derived from 1,2-diacylglycerol intermediates. This notion is supported by the finding that during incubation with radioactively labeled fatty acids the formation of diacylglycero-O-(N,N,N-trimethyl)homoserines parallels the biosynthesis of both diacylglycerophosphocholines and diacylglycerophosphoethanolamines, two classes of phospholipids which are known to be derived from 1,2-diacylglycerols. The mechanism of the formation of the ether bond, however, is as yet unknown. Incubation of C. fusca cultures with 14C-labeled fatty acids or alcohols leads to the formation of fair proportions of wax esters that are labeled in both the acyl and the alkyl moieties, indicating that in these algae fatty acids and alcohols are interconverted. 14C-Labeled long-chain alcohols are not incorporated into the alkyl moieties of ether lipids, whereas labeled 1-O-alkylglycerols are used, though to a very small extent, as precursors of ether phospholipids.

Lipids from callus cultures and suspension cultures of higher plants constitute 5 to 8% of the dry tissue's weight. The predominant lipid classes are the sterols, steryl esters, steryl glycosides and esterified steryl glycosides. Considerable amounts of a variety of sterylglycolipids, whose structures are not completely elucidated, are also present. Triglycerides and phospholipids occur in small proportions, whereas monogalactosyl diglycerides, digalactosyl diglycerides and sulfoquinovosyl diglycerides are present only in traces, if at all. Beta-Sitosterol is the predominant constituent sterol, stigmasterol and campesterol as well as a variety of as yet unidentified sterols occur in smaller proportions. The major constituent fatty acids are palmitic, oleic, linoleic and linolenic acids. Saturated very long-chain fatty acids are found in smaller proportions. Unusual fatty acids, such as epoxy acids, which occur in the seed lipids of certain plants, are not found in tissue cultures derived from these plants. Clucose and traces of galactose are the only sugars obtained by acid hydrolysis of the glycolipids occurring in plant tissue cultures.

Equimolar mixtures of several homologous or vinylogous alkyl methanesulfonates (mesylates) are converted, by a series of reactions, into equimolar mixtures of compounds having one methylene group more, per molecule, than the starting material. Such mixtures can be used for studying the specificity of an enzymatic reaction in a single experiment in vivo or in vitro. The simultaneous synthesis of a mixture of compounds is also of great advantage in the preparation of a series of radioactively labelled substances. It is more convenient and much more efficient to prepare several labelled compounds of a homologous or vinylogous series in a single reaction, and to isolate each of them by chromatographic methods, than to synthesize the individual substances separately.

The lipids of seeds, leaves, and roots of parsley,Petroselinum crispum, and of heterotrophic as well as photomixotrophic cell cultures of this plant were characterized with the aim of finding a system for studying the biosynthesis of unusual fatty acids. It was found that (Z)-6-octadecenoic acid, petroselinic acid, which is the typical constituent fatty acid of triacylglycerols in seeds, occurs only in small proportions, if at all, in leaves, roots, and cell cultures of parsley. In all lipid classes studied petroselinic acid is accompanied by its (Z)-9- and (Z)-11-isomers, oleic and vaccenic acid, respectively. The phosphatidylcholines, phosphatidylethanolamines, and triacylglycerols of both heterotrophic and photomixotrophic callus cultures contain no petroselinic acid but rather oleic and vaccenic acids in equal ratios. Thus, cell cultures of parsley appear to be suitable for studying the biosynthesis of vaccenic acid. The constituent octadecadienoic acids in the lipids of various tissues and cell cultures of parsley consist almost exclusively of the (Z),(Z)-9,12-isomer, linoleic acid, which is derived from oleic acid. (Z),(Z)-6,9- and (Z),(Z)-11,14-Octadecadienoic acids, which could be expected as products of desaturation of petroselinic and vaccenic acids, were not found in any of the lipids of organs and cell cultures investigated.

Resembling the lipids in the leaves and other green organs of intact plants, the lipids in photoautotrophic cell cultures of Chenopodium rubrum were found to contain high proportions of monogalactosyldiacylglycerols and digalactosyldiacylglycerols, as well as fair amounts of sulfoquinovosyldiacylglycerols and diacylglycerophosphoglycerols. Conversely, the heterotrophic cell cultures, from which the photoautotrophic cultures had been derived, contained only traces of these compounds. The heterotrophic cultures were rich in sterols, sterol esters, sterol glycosides, and esterified sterol glycosides. The lipids of photoautotrophic cell cultures contained higher proportions of constituent linolenic acid, but lower concentrations of linoleic acid than those of heterotrophic cultures. In the photoautotrophic cultures, as in green leaves, linolenic acid was predominantly estrified in monogalactosyldiacylglycerols and digalactosyldiacylglycerols. This investigation shows that it is possible to select strains of cell cultures, which are capable of grosing photoautotrophically, with the aim of activating the biosynthesis of specific metabolites.

The chromatographic behavior of some minor components of natural lipids was studied using layers of magnesium oxide and of commercial adsorbents containing magnesium oxide. The lipids investigated included wax esters, cholesteryl esters, diester waxes, and esters, ethers and ether-esters of ethanediol and glycerol. Pronounced differences were found in the patterns of separation of certain lipids on magnesium oxide as compared to silica gel and Florisil. The procedures described afford means for the detection of unusual lipids in natural materials, and their isolation.

The major alkoxylipids of human heart and aorta are alkyl and alk-1-enyl diacyl glycerols, alkyl acyl and alk-1-enyl acyl glycerophosphoryl cholines, and the corresponding glycerophosphoryl ethanolamines. There are no pronounced differences in the composition of corresponding classes of alkoxylipids from heart, aorta, and other human tissues previously reported.

The use of thick layers of adsorbent for the concentration and subsequent isolation of neutral lipid constituents is described. Lipids not perceptible by conventional methods are demonstrated in concentrates of bovine heart extracts and identified as fatty aldehydes, O-alk-1-enyl diglycerides (neutral plasmalogens), and O-alkyl diglycerides (alkoxydiglycerides).

The cleavage of alpha-diols and alpha-amino alcohols with sodium metaperiodate proceeds under mild conditions and in high yields when pyridine is used as reaction medium. The method is suitable for preparative as well as analytical applications.

Procedures are described for the reaction of methanesulfonates with potassium cyanide and for the conversion of the resulting nitriles to methyl esters. Both reactions in this method of chain elongation are quantitative and do not lead to alterations of olefinic double bonds.

Long-chain alkyl and alkenyl bromides are obtained in quantitative yields by the reaction of methane-sulfonates with anhydrous magnesium bromide in absolute ether; cis-trans isomerization of double bonds does not occur.