The separation of phosphatidyl ethanolamine and phosphatidyl serine by column chromatography

A method for the complete separation and apparent quantitative recovery of both phosphatidyl ethanolamine and phosphatidyl serine from lipid extracts of beef brain is presented. Evidence for the purity, quantitative recovery, and the suitability of the technique for the subsequent analysis of the fatty acid and fatty aldehyde composition of the phospholipids is described.     

Separation of milk fat triacylglycerols by argentation thin-layer chromatography

Argentation thin-layer chromatography was investigated as a method of obtaining detailed compositional information about milk fat. A modified argentation thin-layer chromatography procedure, developed to optimize the separation of the complex mixture of total milk fat triacylglycerols, provided nine different groups of triacylglycerols, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester (FAME) analysis was performed to determine the composition of each band. Separation on the basis of chainlength was most pronounced among the fully saturated triacylglycerol groups, as evidenced by the high level of C_4:0 and C_6:0 in bands 7 and 8, respectively. For the cis-monoenoic triacylglycerols, the separation of C_4:0 and C_6:0 was less distinct. The cis,cis dienes and other dienoic, trienoic, or tetraenoic species were principally observed in two bands of retention factor <0.08 on the chromatography plate. Minimal cross-contamination of bands was observed, with the exception of the lowest of the trisaturate bands, band 7, in which trans-monoenes were found to be present. Three samples from different points of the New Zealand dairy season were separated by argentation thin-layer chromatography, and their FAME distributions were measured. In addition to differences in the masses of band extracts from these samples, levels of C_10:0 and C_12:0 in all bands, and levels of monounsaturates in the dienoic and trienoic bands, were found to differ. These changes were generally consistent with a pattern of decreasing fat hardness over the November to March period of a typical dairy season.     

Rapid densitometric determination of triglyceride groups by argentation thin layer chromatography

A method has been developed for the quantitative thin layer chromatographic determination of the triglyceride groups, differing in unsaturation, based on densitometry of the charred bands. Complete separation was achieved by continuous development in an open glass jar. The need for correction coefficients was avoided through addition of bromine to the double bonds prior to charring in the presence of sulphuryl chloride. The accuracy and precision of the method were evaluated on a standard mixture and on sunflower oil, olive oil, lard, cocoa butter, and beef tallow. The method is applicable for triglyceride group analysis of the most common fats and oils which contain saturated, monoene, and diene acids.     

Separation of polyunsaturated fatty acids by argentation thin layer chromatography

A single step, silver nitrate thin layer chromatographic procedure which separates a methyl ester mixture containing 0–6 double bonds is described. Purity of each ester recovered from the silver nitrate plate was 98–99%. Recovery of the esters ranged from 100% for saturates to 77% for pentaenes.     

Detection of adulteration of olive oil by argentation thin layer chromatography

Minute amounts of adulterant seed oils in olive oil can be detected by GLC analysis of fatty acids of the polyunsaturated triglyceride fraction, obtained by TLC on silver nitrate impregnated silica gel. Every possible effort was made to avoid any critical or time consuming manipulation in this method in order to develop it as a routine testing procedure. A complete analysis is possible in less than 2 hr and the detection of as low as 2% adulteration by other seed oils is accurate and reliable.     

Analysis of cereal digalactosyldiacylglycerol molecular species by high-performance liquid chromatography

An optimized procedure for the high-performance liquid chromatographic resolution of molecular species of digalactosyldiacylglycerols from cereals has been developed. The optimal conditions were found by a three-step optimization strategy by taking eluent composition, eluent profile, system temperature, and flow into consideration. Separation of seven molecular species was achieved for oats and wheat-kernel digalactosyldiacylglycerols. Molecular species with the same partition number were eluted in the order of increasing unsaturation when utilizing eluent systems based on alcohols and water. This is reversed as compared to what is found when using eluent systems that contain acetonitrile, in which the elution order of the molecular species with the same partition number is according to decreasing unsaturation.     

The determination of tissue ethanolamine levels by reverse-phase high-performance liquid chromatography

A rapid and sensitive procedure for the determination of ethanolamine levels in mammalian tissues is reported. Ethanolamine was extracted from the tissue with a chloroform/methanol mixture, followed by phase separation. The aqueous phase was subjected to charcoal chromatography and the lluant was derivatized with phenylisothiocyanate. The amount of phenylthiocarbamyl (PTC) ethanolamine in the tissue extract was determined by reverse-phase high-performance liquid chromatography. Quantitation of PTC ethanolamine was linear between 0.1–10 nmol. The pool sizes of ethanolamine in hamster heart, liver and kidney were found to be 1.07, 0.92 and 1.11 μmol/g wet weight, respectively. The sensitivity of the method would allow the determination of ethanolamine in very small tissue samples.     

Quantitative analysis of polyenoic phospholipid molecular species by high performance liquid chromatography

The quantitative analysis of phospholipid molecular species containing polyenoic fatty acids is described. Dinitrobenzoyl derivatives of diacylglycerols prepared from phospholipids were separated into individual molecular species by reversed-phase high performance liquid chromatography (HPLC) using a combination of two solvent systems and were quantified at 254 nm. Thirty-six molecular species were resolved from the phosphatidylcholines of rat hearts, human platelets and Chinese hamster V79-R cells. The derivatives of alkenylacyl molecular species from platelet phosphatidylethanolamine were resolved concomitantly with diacyl molecular species.     

Determination of molecular species of oil triacylglycerols by reversed-phase and chiral-phase high-performance liquid chromatography

A method is described for the determination of molecular species of oil triacylglycerols. The method is based on the analytical separation of the enantiomericsn-1,2-andsn-2,3-diacylglycerols, derived from triacylglycerols, by high-performance liquid-chromatography (HPLC) on a chiral column containing N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(S)-valinecarbonyl-(S)-valine as stationary phase. Model triacyl-glycerol molecules comprising three known fatty acids were isolated from peanut oil and cottonseed oil by a combination of argentation-TLC and reversed-phase HPLC and submitted to partial chemical deacylation. The derivedsn-1,2(2,3)-diacylglycerols were analyzed and fractionated as 3,5-dinitrophenyl urethane derivatives by reversed-phase HPLC according to chainlength and unsaturation. From thesn-1,2(2,3)-diacylglycerol composition and the diacylglycerolsn-1,2-andsn-2,3-enantiomer composition, the individual molecular species of four peanut oil triacylglycerols and one cottonseed oil triacylglycerol were identified and quantitated. The method can be applied to triacylglycerols of any other oil or fat.     

Separation of oxidized and unoxidized molecular species of phosphatidylcholine by high pressure liquid chromatography

Soy phosphatidylcholine (PC) has been separated into its major molecular species by reversephase high pressure liquid chromatography (HPLC). An aqueous methanol gradient was used that allowed detection of the various species by their absorbance at 206 nm. Oxidized species were detected by their absorbance at 234 nm and were resolved from the unoxidized species. This technique has been used to separate and purify unoxidized dilinoleyl phosphatidylcholine (di 18∶2 PC) from other species of soy PC and to monitor the autoxidation of an aqueous suspension of the purified di 18∶2 PC. Two oxidized products were formed from di 18∶2 PC. Further analysis showed that they were PC, but one of the products contained an oxidized and an unoxidized fatty acid; in the other product, both fatty acids were oxidized. Present in part at FASEB 63rd Annual Meeting, Dallas, Texas, April 1979.     

Separation of molecular species ofcis- andtrans-triacylglycerols intrans-hardened confectionery fats by silver-ion high-performance liquid chromatography

Silver-phase high-performance liquid chromatography (HPLC) on silver nitrate-loaded silica achieves incomplete separation of major triacylglycerol (TAG) classes present intrans-hardened fats. The “ChromSpher Lipids” silverloaded cation exchange HPLC column has been found to yield good separations oftrans-hardened TAG, with molecular species well resolved. Separations comparable to those previously possible for nonhardened fats are now possible fortrans-hardened fats. The separation is on the basis of number and type (i.e.cis/trans) of double bonds only; the position of the double bond along the acyl group appears not to influence the separation significantly. The analysis of a palm fraction, hardened to a slip melting point of 37°C and chemically randomized, is presented as an example. This technique offers a new approach to understanding and controlling the hydrogenation and processing oftrans-hardened fats.     

Phosphatidylglycerol molecular species of photosynthetic membranes analyzed by high-performance liquid chromatography: Theoretical considerations

A reversed-phase high-performance liquid chromatography technique was developed to separate, identify, and quantify individual phsphatidylglycerol (PG) molecular species in thylakoid membranes isolated from higher plant leaves. PG was first separated by thin-layer chromatography; then the dinitrobenzoyl derivatives of diacylglycerols produced after phospholipase C hydrolysis of PG were separated by a C₁₈ reversed-phase column and detected at 254 nm. A linear response of the detector was observed in the range of 0.025 to 12 nmol of PG molecular species. It was established that there was an excellent correlation (r=0.996) between the carbon and double-bond number in the aliphatic residues and the relative retention time of dinitrobenzoyl derivatives. A new equivalent carbon number value (ECN^*) which takes into consideration the number ofcis-(n _c ) andtrans-(n _t ) double bonds per molecular species was defined as ECN^*=CN-2n _c -n _t′ where CN is the number of carbon atoms in the aliphatic residues. The logarithm of the retention time increased linearily as a function of ECN^* value. However, in this type of correlation, it may happen that two molecular species of PG having distinct relative retention times has the same ECN^* value. In this case, the two molecular species can be identified by the linear correlation (r=1) existing between the reciprocal of the relative retention time and the number of double bonds (0≤n≤3) in the separate 18:n/Δ3-trans-hexadecenoic acid [16:1(3t) and 18:n/16:0 molecular species series. The advantages of this method are good separation, short elution time, quantitative precision, and predictable retention times of PG molecular species from chloroplast membranes. The method has been used routinely to identify the ten PG molecular species of thylakoid membranes in squash, potato, lettuce, and spinach leaf: 18:3/16:1(3t), 18:3/16:0, 18:2/16:1(3t), 18:2/16:0, 18:1/16:1(3t), 18:1/16:0, 18:0/16:1(3t), 18:0/16:0, 16:0/16:1(3t), and 16:0/16:0.     

Surface characterization of intact fibers by inverse gas chromatography

Inverse gas chromatography (IGC) was used to determine adsorption isotherms and isosteric heats of adsorption for several normal alkanes on a number of intact textile fibers and to determine the specific surface areas of these fibers. Surface areas obtained by IGC were in excellent agreement (except for cotton) with those calculated from the geometric dimensions of circular cross-section fibers and with those obtained by the adsorption of krypton. Heats of adsorption were found to be only slightly higher than the corresponding heats of liquefaction of the probes. The effect of fiber surface purity on adsorption behavior as well as computerized data reduction are briefly discussed.     

Arabidopsis serine decarboxylase mutants implicate the roles of ethanolamine in plant growth and development

Ethanolamine is important for synthesis of choline, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in plants. The latter two phospholipids are the major phospholipids in eukaryotic membranes. In plants, ethanolamine is mainly synthesized directly from serine by serine decarboxylase. Serine decarboxylase is unique to plants and was previously shown to have highly specific activity to l-serine. While serine decarboxylase was biochemically characterized, its functions and importance in plants were not biologically elucidated due to the lack of serine decarboxylase mutants. Here we characterized an Arabidopsis mutant defective in serine decarboxylase, named atsdc-1 (Arabidopsis thaliana serine decarboxylase-1). The atsdc-1 mutants showed necrotic lesions in leaves, multiple inflorescences, sterility in flower, and early flowering in short day conditions. These defects were rescued by ethanolamine application to atsdc-1, suggesting the roles of ethanolamine as well as serine decarboxylase in plant development. In addition, molecular analysis of serine decarboxylase suggests that Arabidopsis serine decarboxylase is cytosol-localized and expressed in all tissue.     

Effect of choline,ethanolamine and serine supplementation on the membrane properties ofMicrosporum gypseum

Phospholipid bases, choline and ethanolamine, when supplemented in the growth medium ofMicrosporum gypseum resulted in an increase in the corresponding phospholipid and total phospholipid content. However, when serine was supplemented, marginal changes were observed. The fatty acid profile of phospholipids remained almost unchanged. The uptake of lysine, leucine and aspartic acid in the spheroplasts of choline- and ethanolamine-grown cells was higher as compared to the control. 1-Anilinonaphathalene-8-sulfonate (ANS) binding to the spheroplast membrane, as calculated from Scatchard plots, demonstrated an increase in the number of binding sites in choline- and ethanolamine-grown cells, while a decrease was observed in the serine-supplemented cells. The results are discussed in terms of the effect of phospholipid polar head group composition on the membrane structure and function of this fungus.     

Fatty acids of serine,ethanolamine, and choline plasmalogens in some marine bivalves

The FA composition of glycerophospholipid (GPL) classes and subclasses was investigated in whole animals of three marine bivalve mollusks: the Japanese oyster Crassostrea gigas, the blue mussel Mytilus edulis, and the Manila clam Ruditapes philippinarum. Individual organs (gills, mantle, foot, siphon, and muscle) of the Manila clam also were examined. The PS plasmalogen (PSplsm), PE plasmalogen (PEplsm), and PC plasmalogen (PCplsm) subclasses were isolated by HPLC, and their individual FA compositions were examined using GC. Plasmalogen forms of PS and PE, when compared to their respective diacyl forms, were found to be specifically enriched with non-methylene-interrupted (NMI) FA (7,15–22∶2, 7, 13–22∶2, and their precursors) and 20∶1n−11 FA. Such a clear specific association was not found for PCplsm. Interestingly, this trend was most apparent in PSplsm, and the above FA were found to be, respectively, the predominant PUFA and monounsaturated FA in the PSplsm isolated from the three species. This specificity was maintained in all the analyzed organs of the Manila clam but varied in proportions: The highest level of plasmalogens, NMI FA, and 20∶1n−11 was measured in gills and the lowest was in muscle. These results represent the first comprehensive report on a FA composition of the PSplsm subclass isolated from mollusks. The fact that NMI FA and 20∶1n−11, which are thought to be biosynthesized FA, were mainly associated with aminophospholipid plasmalogens (PE and PS) is likely to have a functional significance in bivalve membranes.     

Separation of molecular species of ceramides as benzoyl andp-nitrobenzoyl derivatives by high performance liquid chromatography

Ceramides were subjected to direct separation and structural analysis by high performance liquid chromatography after derivatization with benzoyl andp-nitrobenzoyl chloride. The structures of benzoyl andp-nitrobenzoyl derivatives of a representative ceramide, N-heptadecanoyl D-sphingosine, were fully characterized by mass spectrometry. Excellent separation of molecular species of ceramides was achieved with a 10-μ LiChrosorb RP18 column by isocratic elution. Both the acyl group and the long-chain base moiety were separated on this reverse-phase column. The method has been used to determine quantitatively molecular species of nonhydroxy ceramides derived from bovine brain and egg yolk. Comparison of gas liquid chromatographic and high performance liquid chromatographic assays of intact ceramides was made. Presented at the International Society of Fat/ American Oil Chemists' Society in New York, April 29, 1980.     

A mathematical model for the prediction of triglyceride molecular species by high performance liquid chromatography

The physicochemical and theoretical relationships between the traditional equivalent carbon number (ECN) and a proposed elution theory for triglyceride molecular species that has been expressed as a matrix model were demonstrated by multiple regression analysis. It was concluded that the ECN expression has two independent variables, that is, the total acyl carbon number (CN) and the total double bonds (DB), and one dependent variable, the relative retention time (RRT). In the proposed elution equation, there are six independent variables, including the carbon numbers and number of double bonds in each acyl group to be considered.     

Resolution of radiolabeled molecular species of phospholipid in human platelets: Effect of thrombin

Resolution of individual molecular species of human platelet 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines by reverse phase high pressure liquid chromatography (HPLC) allowed a thorough analysis of those phospholipids labeled with [³H]arachidonic acid. Approximately 54% and 16% of the total incorporated radiolabel was found in choline glycerophospholipids and ethanolamine glycerophospholipids, respectively, with ca. 90% of this being found in the 1,2-diacyl molecular species. Eighty percent of [³H]-arachidonic acid incorporated into 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine in resting platelets was equally distributed between 1-palmitoyl-2-arachidonoyl and 2-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, while 70% of the radiolabel in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine was found in 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine. Thrombin stimulation (5 U/ml for 5 min) resulted in deacylation of all 1-acyl-2-[³H]arachidonoyl molecular species of 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-acyl-2-arachidonoyl-sn-glycero-3-ethanolamine. There was also a slight increase in 1-O-alkyl-2-[³H]arachidonoyl-sn-glycero-3-phosphocholine and a significant increase in 1-O-alk-1′-enyl-2-[³H]arachidonoyl-sn-glycero-3-phosphoethanolamine molecular species of over 300%. Thus, HPLC methodology indicates that arachidonoyl-containing molecular species of phosphatidylcholine and phosphatidylethanolamine are the major source of arachidonic acid in thrombin-stimulated human platelets, while certain ether phospholipid molecular species become enriched in arachidonate.