Diglyceride isolation

The quaHty, ie, level of impurities, of the fats and oils used in the manufacture of soap is important in the production of commercial products. Fats and oils are isolated from various animal and vegetable sources and contain different intrinsic impurities. These impurities may include hydrolysis products of the triglyceride, eg, fatty acid and mono/diglycerides proteinaceous materials and particulate dirt, eg, bone meal and various vitamins, pigments, phosphatides, and sterols, ie, cholesterol and tocopherol as weU as less descript odor and color bodies. These impurities affect the physical properties such as odor and color of the fats and oils and can cause additional degradation of the fats and oils upon storage. For commercial soaps, it is desirable to keep these impurities at the absolute minimum for both storage stabiHty and finished product quaHty considerations. 

The isolation of mutants in S. typhimurium by Rick and Osborn (11,12) and mutants in E. coli by Nishijima and Raetz (44) that accumulate the Lipid A precursor indicate that KDO synthesis and Lipid A synthesis are not coordinately controlled. The initial steps in the synthesis of the Lipid A precursor are totally unknown. The temperature sensitive mutants of E. coli isolated by Nishijima and Raetz (44) that are defective in phosphaditylglyc-erol phosphate synthesis at 42°C and accumulate the Lipid A precursors indicate that there is some relationship between the synthesis of phosphatidylglycerol and LPS. The reasons for the acu-cumulation of the Lipid A precursors in this E. coli mutant are not obvious. We have shown that CDP-diglyceride, one of the substrates for phosphatidylglycerol phosphate synthesis, is an inhibitor of D-arabinose-5-phosphate isomerase with an 1 value... 

The simplest type of phosphoinositide is represented by the phosphatide isolated from horse liver or dog liver by McKibbin.171 This consists only of glycerol, myo-inositol, phosphoric acid, and fatty acids it probably has structure LXXX. Similar inositides, differing only in the nature of the fatty acids, have been isolated from wheat germ,187 beef heart, 188 and beef liver.189 That the major portion of the fatty acids is attached to glycerol, not to myo-inositol, was shown by the isolation of a diglyceride on mild hydrolysis.189... 

Although the occurrence of analogs of PAF in mammalian cells was always considered a possibility, Mueller et al. (1984) first reported the presence of 1-0 long-chain acyl-2-acetyl-glycero-3-phosphocholine in rabbit neutrophils. Later, Tokumura et al. (1989) isolated a vasopressor active phosphatidylcholine fraction from bovine brain. Treatment of the fraction with phospholipase C yielded a diglyceride component which was converted to the t-butyldimethylsilyl derivative. Analysis of the latter by gas-liquid chroma-... 

By the method of Hanahan (hydrolysis with 2 N hydrochloric acid for 20 min. at 100°), a diglyceride was isolated this proves that two fatty acids of the phospholipid are both linked to glycerol. 

While it may be true that SC-CO2 can effect little separation of mono- and diglycerides, we have found it to be useful in isolating unreacted esters and triglycerides from our crude mixtures. Table II provides a summary of experimental conditions and results for the fractionation of a crude tri- EPA reaction mixture. 

A crude enzyme mixture was isolated from the fermentate of Candida rugosa (ATCC No. 14,830), which is reported to produce high activity lipases ( ). The enzyme mixture was added to a 20% butterfat emulsion. A cheese-like flavor developed after 3 hours of incubation at 37°C. A desirable Romano cheese note developed after continued incubation at room temperature for three days. Nelson (6) studied lipolyzed butterfat flavor and concluded that the surface active characteristics of both fatty acids and mono- and diglycerides were important in the lipolyzed system. A tempering period of hours or even days was usually required to establish equilibrium at the interface of aqueous and fat phases. He pointed out that the lipolyzed flavor appeared to intensify as the equilibration proceeded and that this intensification was sometimes mistaken for residual lipolytic activity. 

Seven different classes of lipids, including fatty acids, phospholipids, cholesteryl ester, cholesterol, triglycerides, diglycerides, and monoglycerides are isolated and separated (Fig. 8.12). Fatty acids and phospholipids are both... 

The isolated brush border vesicles from the plasma membrane of the microvilU is the simplest in vitro system used so far. The interaction of lipid with rabbit intestinal brush border vesicles has been investigated by Proulx et al. [50] who found that PC, phosphatidylethanolamine, cholesterol, diglyceride as well as fatty acids were taken up by vesicles. Barsukov et al. [51] have shown that transfer of PC from PC vesicles to isolated brush border vesicles can occur in the presence of PC-exchange protein. The use of brush border vesicles is an interesting new approach that permits detailed studies of rate of transfer of specific lipids into the plasma membrane of the enterocyte. The model is seriously hmited by the fact that incubation with solutions containing bile salts at a concentration above the critical micellar concentration will result in partial or total solubilization of the membrane vesicles. 

Sulpholipids of various chemical composition have been isolated from diatoms (e.g. Anderson et al., 1975). Mono- and polyester glycosyl sulphates or phosphate diglycerides account for a group of polar lipids which are found in large amounts in certain Fucaceae (Liem and Laur, 1976). 

Daleo, G.R. Piras, M.M. Piras, R. Diglyceride kinase activity of microtubules. Characterization and comparison with the protein kinase and ATPase activities associated with vinblastine-isolated tubulin of chick embryonic muscles. Eur. J. Biochem., 68, 339-346 (1976)... 

The ability of PI synthetase to use 5-deoxy-5-fluoro-myo-inositol (4) as a substrate was confirmed by use of a radiolabeled compounds as shown in Figure 7. PI synthetase incorporated the analog into lipid in a time-dependent manner. The incorporation was absolutely dependent on the presence of CDP-diglyceride and was inhibited by the presence of myo-inositol (1) in the incubation mixture, as expected for PI synthetase. Chromatography of the reaction mixture revealed that a single radiolabeled product was formed with a mobility similar to, but distinct from, that of PI. Subsequent analysis has shown that the product is converted to a water-soluble form on mild alkaline hydrolysis and yields 5-deoxy-5-fluoro-myo-inositol (4) on treatment with phospholipase D, in agreement with the formation of phosphatidyl-5-deoxy-5-fluoro-myo-inositol as the product (data not shown). Determination of the absolute structure of these phospholipids awaits large-scale enzymatic synthesis, isolation of the product, and studies by mass spectrometry and NMR spectroscopy. 

This has enabled neutral plasmalogens to be detected in the presence of free aldehydes [183] combined and free aldehydes to be separated [183] and aldehydes, dimethylacetals and methyl esters to be fractionated and isolated [185] (see procedures, p. 373). Neutral plasmalogens can be detected by means of the SRS-technique (p. 88) after chromatographing in the first direction, the alkenyl ethers are hydrolysed on the layer with hydrogen chloride vapour the aldehydes and the diglycerides formed are then separated by chromatography in the second direction [183]. Fig. 134 illustrates the detection of neutral plasmalogens in a shark liver oil by means of TLC. 

Two groups have reported that lipid extracts of mouse tumours contain compounds which riiigrate ahead of the triglyceride fractions on adsorbent layers [113, 191] these substances were considered at first to be methyl esters of fatty acids but more detailed studies of the material isolated through TLC, showed that they were alkyl diglycerides [191]. 

Fig. 136. Isolation of neutral plasmalogens and alkyl diglycerides from the human perinephrium [181]. Adsorbent silica gel G solvent hexane-diethyl ether (99 + 5), double development times of run 40 min each visualisation carbonisation by heating with chromic acid/sulphuric acid, a lipid extract 6 first concentration stage c second concentration stage d third concentration stage e neutral plasmalogens / alkyl diglycerides...

Furthermore it was possible to demonstrate the presence in the intestine of all the enzymes required for these steps. Fatty acid-coenzyme A synthesis was demonstrated by Ailhatjd et al. (1962) and by Clark and Hubscher (1960). The involvement of phosphatidic acid could be demonstrated by isolation of radioactive phosphatidic acid in intestinal preparations incubated with radioactive fatty acids or phosphoric acid (Johnston and Bearden 1960). Furthermore, a significant dilution effect in the synthesis of glycerides from C-fatty acids occurred by the addition of phosphatidic acid (Clark and Hubscher 1961). Phosphatichc acid phosphatase activity in the intestine was reported by Coleman and Hubscher (1962) and by Johnston and Bearden (1962). Finally, the conversion of diglyceride into triglycerides by the addition of a fatty acid — Co A was shown to take place in the intestine by Clark and Hubscher (1961). 

Figure 8. The nomographs for the determination of 1 % (a) or 10 % (b) residual activities of various enzymes. POD (I) represents isolated peroxidase POD (S), peroxidase in suspension MGLase, monoglyceride lipase DGLase, diglyceride lipase PL-C, phospholipase C PL-D, phospholipase D LCase, lecithinase and Vit. C 90 %, vitamine C as a control for the retention of 90 % active form.

Gadner HW. Preparative isolation of monogalactosyl and digalactosyl diglycerides by thin layer chromatography. J Lipid Res 1968 9 139-41. 

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