Biomedical subjects
Publications and source records attributed to W W Christie.
A combination of hplc in the silver ion mode and gc-ms of picolinyl ester derivatives was used to identify the fatty acids in a fresh-water mussel, Unio tumidus, from the Danube in Bulgaria. A number of novel fatty acids were found, including 14-methylpentadec-6-enoic and 17-methyloctadec-8-enoic acids. Eicos-7-enoic acid was a major component.
Two methods for the separation of cholesterol esters, based on the number of double bonds in their fatty acid moieties, are presented. Silver ion chromatography, usually performed on thin-layer chromatographic plates, was made suitable for high-performance liquid chromatography (HPLC) and solid-phase extraction. Separation on a bonded sulphonic acid phase loaded with silver ions was achieved with cholesterol esters containing up to six double bonds in their fatty acid moieties. No cross-contamination between fractions with different numbers of double bonds was detected with the HPLC method, was demonstrated by subsequent gas chromatographic analysis of the fatty acid moieties, following transmethylation. For adequate separations with the solid-phase extraction columns it proved important to avoid overloading. The methods may be of use for the off-line analyses of the sterol compositions of the isolated fractions, which each contain sterol esters with an equal number of double bonds in their fatty acid moieties.
We report here the finding that normal, young cartilages, in distinction from all other tissues examined, have unusually high levels of n-9 eicosatrienoic (20:3 cis-delta 5,8,11) acid and low levels of n-6 polyunsaturated fatty acids (n-6 PUFA). This pattern is identical to that found in tissues of animals subjected to prolonged depletion of nutritionally essential n-6 polyunsaturated fatty acids (EFA). This apparent deficiency is consistently observed in cartilage of all species so far studied (young chicken, fetal calf, newborn pig, rabbit, and human), even though levels of n-6 PUFA in blood and all other tissues is normal. The n-9 20:3 acid is particularly abundant in phosphatidylethanolamine, phosphatidylinositol, and the free fatty acid fractions from the young cartilage. Several factors appear to contribute to the reduction in n-6 PUFA and the appearance of high levels of the n-9 20:3 acid in cartilage: 1) limited access to nutritional sources of EFA due to the impermeability and avascularity of cartilage, 2) rapid metabolism of n-6 PUFA to prostanoids by chondrocytes, and 3) a unique fatty acid metabolism by cartilage. Evidence is presented that each of these factors contributes. Previously, EFA deficiency has been shown to greatly suppress the inflammatory response of leukocytes and rejection of tissues transplanted into allogeneic recipients. Because eicosanoids, which are derived from EFA, have been implicated in the inflammatory responses associated with arthritic disease, reduction of n-6 PUFA and accumulation of the n-9 20:3 acid in cartilage may be important for maintaining normal cartilage structure.
(1) Rat and ovine hepatocytes were incubated in monolayer culture with various fatty acids to determine their effects on the composition of the lysophosphatidylcholine that was secreted. (2) No lysophosphatidylcholine was detected in the medium from the ovine hepatocytes even though these cells were hormonally responsive and they secreted phosphatidylcholine and triacylglycerol in very-low-density lipoprotein. (3) Lysophosphatidylcholine was readily detected in the incubation medium of rat hepatocytes. The predominant fatty acids in this lipid were unsaturated. Stearate and arachidonate contributed 15 and 34%, and 24 and 26% of the total fatty acids when hepatocytes from male and female rats were used, respectively. The relative proportions of stearate and arachidonate in the phosphatidylcholine secreted from the hepatocytes were 20 and 14%, and 28 and 21% for the males and females, respectively. The equivalent values for stearate and arachidonate for phosphatidylcholine in the hepatocytes were 18 and 17% and 33 and 22% for male and female rats. These results provide further indications of sex differences in hepatic phospholipid metabolism and extend this to the secretion of phosphatidylcholine and lysophosphatidylcholine. (4) The addition of 1 mM stearate to the incubation medium did not significantly decrease the proportion of arachidonate in the lysophosphatidylcholine obtained from the hepatocytes of the male rats. However, the relative proportion of arachidonate was decreased in incubations that contained 1 mM oleate or linoleate. (5) The results provide evidence that the preferential secretion of unsaturated lysophosphatidylcholine by the liver may provide a system for transporting unsaturated fatty acids and choline to other organs in non-ruminant animals. However, this mechanism may not operate for ruminants.
The fatty acid content of triacylglycerols (TG) in arterial and mammary venous blood plasma has been analysed in five cows that were close to peak lactation. The fatty acids in arterial TG were extracted by the mammary gland in amounts 14:0 less than 16:0 less than 18:0 and 18:0 greater than 18:1n - 9 greater than 18:2. The difference of extraction between palmitate and stearate was similar in different TG species. The fatty acid 18:1n - 7, which was largely the trans isomer, was extracted more than 18:1n - 9. Significant amounts of phytanic acid were acylated in plasma TG, but not extracted by the mammary gland.
Diacylglycerols have been separated as their (S)-(+)-or (R)-(-)-1-(1-naphthyl)ethyl urethanes by high performance liquid chromatography (HPLC) on a column of silica gel with 0.5% 2-propanol in hexane as the mobile phase. The elution order of components derivatized with the (S)-form of the reagent was 1,3-, followed by 1,2-, and finally 2,3-diacyl-sn-glycerols. The elution order of 1,2- and 2,3-diasteromers was reversed when the (R)-form of 1-(1-naphthyl)ethyl isocyanate was used for derivatization. Single-acid 1,2- and 2,3-diastereomers were separated to the baseline with a resolution factor from 5.2-5.7, and the resolution factor between 1,3- and 1,2- or 2,3-diacyl-sn-glycerol derivatives was more than 23. Molecular species of single-acid diacylglycerol derivatives were separated in the sequence 18:1 less than 18:0 less than 18:2 less than 16.0. In order to assess this methodology as part of a procedure for the stereospecific analysis of triacyl-sn-glycerols, we prepared diacyl-rac-glycerols from maize oil, evening primrose oil and egg yolk triacylglycerols by partial hydrolysis with ethyl magnesium bromide. The 1,3-, 1,2- and 2,3-diacyl-sn-glycerols as (S)-(+)-1-(1-naphthyl)ethyl urethanes were isolated and their fatty acid compositions were determined. Although this only permitted an indirect determination of the compositions of positions sn-1, -2 and -3, it was sufficient to indicate the potential of the methodology because results comparable to those published earlier were achieved.
The fatty acids of seed oils of the Flacourtiaceae, Hydnocarpus anthelmintica, Caloncoba echinata and Taraktogenus kurzii, have been examined by a combination of capillary gas chromatography, silver ion high performance liquid chromatography and gas chromatography-mass spectrometry. In addition to the common range of cyclopentenyl fatty acids found in such oils, 13-cyclopent-2-enyltridec-4-enoic acid was a major component of H. anthelmintica and was identified by mass spectrometry as its picolinyl ester and dimethyldisulphide adduct. It has not previously been found in nature. In the other seed oils, the isolated double bond in the corresponding fatty acid was in position 6, as expected. Similarly, cis-4-hexadecenoic acid and C16 and C18 cyclopentyl fatty acids were identified for the first time in H. anthelmintica. Iso- and anteiso-methylbranched fatty acids were present in trace amounts.
Commercial solid-phase extraction columns packed with a stationary phase with bonded benzenesulfonic acid groups are readily converted to the silver ion form and can then be used for silver ion chromatography of lipids. To illustrate the utility of such procedures, methyl ester derivatives of fatty acids with zero to six double bonds were separated from each other by a simple stepwise elution scheme. -Christie, W. W. Silver ion chromatography using solid-phase extraction columns packed with a bonded-sulfonic acid phase.
Molecular species representative of the wide range of triacylglycerols occurring in nature, ranging from relatively saturated fats such as that from sheep adipose tissue through polyunsaturated seed oils, including sunflower and linseed oils, to an oligounsaturated fish oil, have been resolved by high-performance liquid chromatography in the silver ion mode. The stationary phase consisted of an ion-exchange medium, which was a silica gel matrix with bonded sulphonic acid moieties, loaded with silver ions. The mobile phase for the more saturated fractions was a gradient of acetone into 1,2-dichloroethane-dichloromethane, then acetonitrile was introduced to elute polyunsaturated fractions. A mass detector was employed to monitor separations. Fractions were collected via a stream-splitter for identification and quantification by gas chromatography as methyl esters. Excellent resolution was obtained on the silver ion column with no contamination of fractions with silver ions, while the column was stable and retained its activity in prolonged use.
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Rat adipocytes were used in vitro to compare the positional distributions of fatty acids of intra- and extra-cellular origin in triacyl-sn-glycerols. Fatty acids of extracellular origin were esterified to each position in similar, but not identical, proportions to the natural distributions. A high proportion of the oleic acid synthesised in the tissue by desaturation of exogenous stearic acid was found in position sn-3. When palmitic acid was the only fatty acid added, tripalmitoylglycerol was synthesised by the adipocytes. The rate and pattern of fatty acids synthesised de novo from acetate was dependent on the age of the donor rat and the concentration of acetate and presence or absence of long-chain fatty acids in the medium. The newly synthesised fatty acids were esterified in very different proportions from the natural distributions and thus from those of extracellular fatty acids. The results are discussed in terms of esterification of the fatty acids from the two sources in different compartments of the cell.
Rat adipocytes were used in vivo to compare the esterification of exogenous fatty acids and fatty acids formed de novo from glucose or acetate. Pure single fatty acids added to the medium were esterified at comparable rates but marked differences were observed when the same acids were supplied as components of a fatty acid mixture of a composition similar to that in the tissue. Fatty acids synthesised de novo from acetate by adipocytes in a medium containing high concentrations of acetate were located predominantly in diacylglycerols. The effect was most marked with adipocytes from older rats and was enhanced by the presence of exogenous long-chain fatty acids. Exogenous oleic acid was esterified predominantly into triacylglycerols at all concentrations of acetate. No such accumulation of endogenously-synthesised fatty acids in diacylglycerols occurred when glucose was the precursor for fatty acid synthesis. The diacylglycerols formed were almost entirely of the sn-1,2-configuration.
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