Methodology for the separation o plant lipids and application to spinach leaf and chloroplast lamellae

Procedures for separation of complex plant lipids and results obtained are reviewed. Procedures based on DEAE cellulose and silicic acid chromatography, which may be preceded by countercurrent distribution, are presented for separation of the individual glyceroland sphingolipid classes of spinach leaf and chloroplast lamellae. These procedures appear to be generally applicable to photosynthetic tissue of plants and algae. The separation and infrared spectra of mono-and digalactosyl diglycerides, lecithin, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl inositol, plant sulfolipid, cerebroside, and sterol glycosides from spinach are recorded. Chloroplast lamellae lipids are in the molar ratio monogalactosyl diglyceride (14.0), digalactosyl diglyceride (8.0), phosphatidyl glycerol (5.5), sulfolipid (3.9), lecithin (2.0), phosphatidyl inositol (1.0). Phosphatidyl ethanolamine, cerebrosides, and sterol glycosides were not detected in chloroplast lamellae. Fatty acid composition of individual lamellae lipids have been determined: The galactosyl lipids contain more than 90% trienoic acids.Trans-3- hexadecenoic acid is restricted almost exclusively to phosphatidyl glycerol. In this report techniques which have been applied to the isolation of plant glycero- and sphingolipids are reviewed and a new scheme presented for the separation of several of the plant lipid classes. Results obtained with spinach leaf and its photosynthetic apparatus are presented and discussed. Paper III in the series Plant and Chloroplast Lipids. Presented at the 15th Annual Summer Program, “Symposium on Quantitative Methodology in Lipid Research”, Aug. 3–7, 1964. Literature is reviewed to July 1964.     

Lipid composition of beef and human pituitary glands

The lipid composition of beef and human pituitary was determined by chromatographic and spectrophotometric methods. Beef pituitary lipid contained about 25% nonpolar lipids and 75% phospholipids whereas nonpolar lipids made up approximately 60% of the total in human pituitaries. The main nonpolar (i.e., low polarity) lipids in human pituitary were triglycerides, cholesterol, free fatty acids and an unidentified component in the triglyceride fraction. Cholesterol was the major nonpolar lipid component in freshly collected beef anterior and posterior pituitary, but the amount of free fatty acids appeared to increase during storage. Preliminary investigation of the unknown nonpolar lipid in human pituitaries suggested that it was an unsaturated hydroxy compound with no carbonyl functions. Thin layer chromatography indicated that it was also present in smaller amounts in freshly collected beef pituitaries. The main phospholipids of beef anterior, posterior and human pituitary were phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl inositol, phosphatidyl serine and sphingomyelin. The fatty acid composition of total nonpolar lipids, free fatty acids, total phospholipids, phosphatidyl ethanolamine and phosphatidyl choline of beef anterior and posterior pituitary was determined by gas liquid chromatography. Mixtures of saturated and unsaturated fatty acids ranging from C₁₂ to C₂₂ were present; the main fatty acids were palmitic, stearic, oleic, linoleic and arachidonic.     

Postnatal changes in the phospholipid composition of livers from young lambs

The total lipids were extracted from the livers of newborn lambs, from the livers of lambs during the first week after birth and from the livers of adult sheep. After separation from the nonphospholipids on columns of silicic acid the phospholipids were analyzed by thin layer chromatography and quantitative gas liquid chromatography. In all samples phosphatidyl choline and phosphatidyl ethanolamine together accounted for about 80% of the total liver phospholipids. The phosphatidyl choline-phosphatidyl ethanolamine ratio in the livers of the newborn lambs was markedly less than the ratio in the livers of the adult sheep. Moreover there was a pronounced increase in the phosphatidyl cholinephosphatidyl ethanolamine ratio in the livers of the lambs during the first week after birth. In the liver phospholipids of the lambs the concentration of phosphatidyl inositol was lower and the concentrations of phosphatidyl serine and sphingomyelin were greater than the corresponding concentrations in the liver phospholipids of the adult sheep. It is proposed that the change in the phosphatidyl choline-phosphatidyl ethanolamine ratio in the livers of the lambs during the first week after birth is due, at least in part, to the marked change that occurs in the linoleic acid-arachidonic acid ratio in the tissues of the lamb during this period.     

An unsaturated phosphonic acid analogue of phosphatidylethanolamine and its activity in blood-clotting systems

An unsaturated phosphonolipid analogous to phosphatidylethanolamine,rac-dioleoylglyceryl(2-aminoethyl)phosphonate, was synthesized by a general method introduced by Baer for similar saturated substances. An improvement was made in the preparation of the phthalimidoethyl-phosphonic acid precursor. The phosphonolipid was purified by DEAE cellulose and silicic acid chromatography. It was tested by comparison with synthetic phosphatidyl (dioleoyl) ethanolamine and phosphatidyl(dilinoleoyl) ethanolamine in the Hicks-Pitney test and in a test for prothrombin conversion by using purified blood coagulation factors. In both tests it had more acceleratory activity than the synthetic phosphatidylethanolamines.     

Analytical fractionation of complex lipid mixtures: DEAE cellulose column chromatography combined with quantitative thin layer chromatography

A quantitative chromatographic procedure for the fractionation of complex lipid mixtures is described. The method utilizes diethylaminoethyl (DEAE) cellulose column chromatography followed by thin layer chromatography (TLC). Spots produced in TLC are charred with sulfuric acid-potassium dichromate and heat and are then measured by quantitative densitometry. Results obtained with beef brain and beef heart mitochondrial lipids are presented, and the close correspondence between column isolation procedures and the new procedure is demonstrated. Methods utilizing only column chromatography, column chromatography and TLC, and one- and two-dimensional TLC without column chromatography are compared.     

Phospholipids of human serum

Phospholipids extracted from normal human serum were fractionated into lecithin, lysolecithin, sphingomyelin, phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, phosphatidyl serine, and phosphatidyl inositol. Identification of each was established by thin-layer chromatography and infrared spectrophotometry. The content of plasmalogen was determined in both lecithin and phosphatidyl ethanolamine fractions. The composition of fatty acids and fatty aldehydes in isolated phospholipids is presented. The degree of unsaturation as reflected in the average content of double bonds per molecule of the fatty acids in phospholipids was: lecithin 1.2, choline plasmalogen 2.1, lysolecithin 0.6, sphingomyelin 0.2, phosphatidyl ethanolamine 2.8, lysophosphatidyl ethanolamine 1.0, phosphatidyl serine 1.0, and phosphatidyl inositol 1.8. Both chlline and ethanolamine plasmalogen aldehydes were predominantly saturated. Molecular weight of each phospholipid was calculated from determined fatty acid and fatty aldehyde compositions; the phosphorus factor for each phospholipid was computed. On a weight percent basis, lecithin, sphingomyelin, and lysolecithin accounted for 95% of the total phospholipids. The ethanolamine-containing phospholipids accounted for 2.5%, and the remainder was divided among phosphatidyl inositol, choline plasmalogen and phosphatidyl serine. Presented in part at the AOCS Meeting, Houston, April, 1965. Dept. of Health, Education and Welfare, USPHS.     

Determination of polar lipids: Quantitative column and thin-layer chromatography

The structures of the polar lipid classes of plants and animals are presented, their nomenclature discussed, and suggestions are presented for clarification of nomenclature. The three general types of quantitative chromatographic procedures (column chromatography, thin-layer chromatography, and combinations of column and thin-layer chromatography) available for polar lipids are reviewed and a new quantitative two-dimensional thin-layer chromatographic procedure is presented. Useful quantitative procedures employing columns of cellulose, silicic acid, silicic acid mixed with silicate, magnesium silicate, and ion exchange celluloses are presented. New findings with diethylaminoethyl cellulose columns are described. New quantitative procedures employing silicic acid, magnesium silicate, or diethylaminoethyl cellulose column chromatography with quantitative thin-layer chromatography are described.     

Main constituents of rapeseed lecithin

Commercial rapeseed lecithin has been analyzed after separation by silicic acid column chromatography. Besides neutral oil (40%), four major constituents have been found, viz., phosphatidyl ethanolamine (18%), phosphatidyl inositol (8%), phosphatidyl choline (16%) and sterol glycosides (8%). Among the minor fractions lysophosphatidyl ethanolamine accounts for about 2%. The phosphatides are characterized by low erucic acid content and the major fatty acids are palmitic, oleic and linoleic acids.     

Lipid class and fatty acid composition of intact peripheral nerve and during wallerian degeneration

Lipid extracts from normal cat, chicken, and beef sciatic nerve were fractionated into their components by combinations of silicic acid, Florisil, DEAE-cellulose, or silicic acid-silicate column chromatography. The constitutent fatty acids of total lipid extracts and of individual lipid classes were qualitatively and quantitatively determined as their methyl esters by gas chromatography. These methods were also applied to lipid extracts from cat sciatic nerve undergoing Wallerian degeneration at 8, 16, 32, and 96 days after section and to chicken sciatic nerve undergoing demyelination due to organophosphate poisoning. All fatty acids were markedly decreased in the total lipids of cat sciatic nerve at 96 days after section and most of these were decreased at 32 days. As early as 8 days after section 16:0, 16:1, 18:2, 20:0, and 20:4 showed decreases, while 18:0, 18:1, 22:1, 22:5, 22:6, and 24:1 did not begin to show decreases until 16 days after section. The decreases in fatty acids were considered to be due to increased catabolism, decreased synthesis, or increased removal of fatty acids from nervous tissue. The fatty acid content of the total lipids of chicken nerve undergoing demyelination resembled that of cat sciatic nerve between 16 and 32 days after section. Myelin lipids, sphingomyelin, cerebrosides, and phosphatidyl ethanolamine (PE) began to decrease as early as 8 days after section in cat sciatic nerve. Phosphatidyl serine (PS) also decreased at this time. Cholesterol, lecithin, and ethanolamine plasmalogen did not begin to decrease until 16 days after section and phosphatidyl inositol (PI) did not decrease until 32 days after section. Triglycerides decreased markedly at 8 days after section gradually returning to normal by 96 days. This was accompanied by a transient increase in free fatty acids and monoglycerides. Cholesterol esters and lysolecithin increased markedly at 8 days after section and were higher than normal levels even at 96 days after section. In chicken sciatic nerve undergoing demyelination after organophosphate poisoning, cerebroside was the only myelin lipid which decreased in amt, while cholesterol esters and diglycerides increased. Sphingomyelin and cerebrosides containing 16:0, 18:0, 18:1, 18:2, 20:0, 22:0, 23:0, 24:0, 24:1 seemed to be most susceptible to degradation or interference in synthesis in degenerating nerve. For the most part, these fatty acids were observed to increase in cholesterol esters, free fatty acids, and, in some instances, triglycerides. The changes in various lipid classes and their constituent fatty acids are discussed in relation to various cellular changes which accompany degeneration.     

Lipid composition of beef heart ventricle

The lipid class composition of beef heart ventricle was determined by a combination of diethylaminoethyl cellulose column chromatography and quantitative thin-layer chromatography. Percentages of the total lipid were: triglyceride, 43.6; cholesterol, 7.4; phosphatidyl choline, 22.8; phingomyelin, 4.0; phosphatidyl ethanolamine, 11.2; diphosphatidyl glycerol, 5.8; phosphatidyl serine, 1.2; phosphatidyl inositol, 3.0; phosphatidyl glycerol, 0.9; unsaturated hydrocarbon, 0.02; saturated hydrocarbon, 0.20. Nonlipid components represented 33.6% of the crude chloroform/methanol extracts.     

Lipids of subcellular particles

Methods for isolation and characterization of subcellular particles as well as procedures for analysis of lipid class composition are discussed. The literature on distribution of lipids in subcellular particles is then reviewed. Pertinent new data from our laboratories are presented as well. The isolated particles are related to the organelles to which they correspond in the cell and are discussed with regard to heterogeneity and morphological integrity. Confusion can arise with regard to subcellular particles unless it is appreciated that: 1) preparation of particles of high purity generally requires more than the classical differential centrifugation scheme (both differential and gradient centrifugation may be required); 2) it is hazardous to apply exactly the same procedure for all tissues; 3) all subcellular fractions must be thoroughly characterized. The more recently devised DEAE cellulose column and thin-layer chromatographic procedures for analysis of lipid class composition are more reliable than the older hydrolytic or silicie acid column or paper chromatographic techniques. The chief lipid components of mitochondria from all organs and species are lecithin, phosphatidyl ethanolamine, and cardiolipin (diphosphatidyl glycerol). Despite the fact that reports in the literature are in agreement that phosphatidyl inositol is a major component of mitochondria, it is concluded on the basis of new data obtained from highly purified mitochondria and improved analytical methods that phosphatidyl inositol is not a major component of mitochondria. The presence of a relatively large amount of phosphatidyl inositol in mitochondrial preparations is probably related in part to contamination with other particles. Some analytical procedures are demonstrated to give erroneous values for this lipid class. It is also concluded that phosphatidyl serine, phosphatidic acid, sphingomyelin, cerebrosides, and lysophosphatides, reported to occur in mitochondria, are not characteristic mitochondrial components and furthermore that the large amount of uncharacterized mitochondrial phospholipid reported is actually an analytical artifact. Microsomes appear to be similar to mitochondria except that cardiolipin is either low in or absent from microsomes. Available data indicate nuclei to be rather similar to mitochondria and microsomes, at least in some organs. Studies of the fatty acids of subcellular particles indicate that different particles from one organ have very similar fatty acid compositions. It is clear that there are marked variations in fatty acid composition of particles from different organs and from different species. Differences in dietary fat may be associated with marked changes in fatty acid composition, although brain mitochondrial lipids are largely unchanged. Each lipid class from mitochondria of most organs appears to have a fairly characteristic fatty acid composition. Cardiolipin from some organs contains primarily linoleic acid, phosphatidyl ethanolamine contains large amounts of linoleic and higher polyunsaturates, and lecithin is similar to phosphatidyl ethanolamine except that it does not contain as much arachidonic acid and/or other highly unsaturated fatty acids. New data, the first to be reported, are presented for heart mitochondrial cardiolipin, phosphatidyl ethanolamine, and lecithin. It is concluded that there are two basically different types of membranous structures. Myelin is the chief representative of the metabolically stable type of membrane structure while mitochondria represent the more labile type. The two types of membranes have very different in vivo properties and very different lipid compositions. Myelin is characterized by a high content of cholesterol and sphingolipids with more long chain saturated or monoenoic fatty acids while mitochondria are characterized by a low cholesterol content, little or no sphingolipid, and highly unsaturated fatty acids. It is clear that formulations of the myelin type membrane structure such as that of Vandenheuvel cannot apply to mitochondria. It is postulated that membrane structures intermediate between the extremes represented by myelin and mitochondria exist.     

Composition and structure of phospholipids in chicken muscle tissues

Lipids extracted from breast muscle and thigh muscle of one-year old chickens on a standard MSU-Z-4 diet have been fractionated by silicic acid column chromatography into nonphospholipids, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl choline (lecithin), and sphingomyelins. Phospholipid fractions were identified by thin-layer chromatography and the quantity of each determined by gravimetric analysis, analysis of the phosphorus content, and infrared spectra. The phospholipid content of thigh muscle (dark meat) lipids was higher than that in the breast muscle (white meat). Phosphatidyl choline and phosphatidyl ethanolamine were found in relatively greater amts than phosphatidyl serine and sphingomyelins. Enzymatic hydrolysis followed by gas-liquid chromatographic analysis of the fatty acids liberated and those in the lysocompounds was used to establish the positional specificity of the fatty acids in the phosphoglycerides. The polyunsaturated fatty acids are located primarily at the β-position and the saturated fatty acids at the α′-position. The qualitative and quantiative determination of the plasmalogens was also accomplished. Michigan Agriculture Experiment Station Journal Article No. 3527. Presented at the AOCS Meeting in Chicago, October, 1964.     

Phospholipids of the sea anemone: Quantitative distribution; absence of carbon-phosphorus linkages in glycerol phospholipids; structural elucidation of ceramide aminoethylphosphonate

The phospholipid composition of the sea anemone (Anthopleura elegantissima) was determined by quantitative thin-layer chromatography (TLC). Phosphonic acids were not detected in phosphatidyl choline, phosphatidyl ethanolamine, or phosphatidyl serine following isolation and hydrolysis. The structure of ceramide aminoethylphosphonate, a phosphonolipid, was elucidated by hydrolysis and oxidative degradation followed by characterization of the products by TLC and gas-liquid chromatography. The long-chain base was shown to be sphingosine with the 2-aminoethylphosphonic acid group attached through the primary hydroxyl group at carbon one. Ceramide aminoethylphosphonate is therefore 1-(O-phosphonoethylamine)-N-acyl sphingosine.     

Lipid metabolism ofAgaricus bisporus (Lange) sing.: I. Analysis of sporophore and mycelial lipids

The lipid components of four strains ofAgricus bisporus (Lange) Sing., the cultivated mushroom, were analyzed. Both sporophore and mycelial samples were obtained from beds in normal production. A method for obtaining mycelium free of compost was developed. Neutral lipids were separated from polar lipids by silicic acid column chromatography. Each fraction was separated by thin layer chromatography. Fatty acid methyl esters were analyzed by gas liquid chromatography and mass spectrometry. Sporophore extracts contained free sterol, free fatty acid, triglycerides, phosphatidyl choline and phosphatidyl ethanolamine. High amounts of linoleic acid were found in both neutral and polar lipid fractions. Mycelial extracts contained free fatty acids, triglycerides, phosphatidylcholine and phosphatidyl ethanolamine. No free sterol could be detected. Linoleic acid was also present in large amounts. Paper 3798 in the Journal Series of The Pennsylvania Agricultural Experiment Station.     

Fatty acids in phospholipids isolated from human red cells

Total lipids of packed erythrocytes from healthy men 22 to 25 years old were extracted with chloroform-methanol mixture. Phospholipid classes were separated from neutral lipids and pigments on a silicic acid column. Phosphatidyl inositol (PI) was freed of its contaminants phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS) on an aluminum oxide column. Additional silicic acid columns with modified solvent systems were needed for complete separation of other overlapped phospholipid classes. The identification of phospholipids in each eluted fraction was accomplished by TLC, using the appropriate spray tests and reference compounds, and confirmed on each of the isolated phospholipids by IR spectrophotometry. The total content of phospholipids as determined by phosphorus analysis was found to be 2.63 mg/ml of packed cells. These phospholipids were found to have the following composition (in per cent of total phospholipid): PI, 2.3; PE, 13.4; ethanolamine plasmalogen (EP), 14.5; PS, 3.9; lecithin (L), 34.2; choline plasmalogen (CP), 1.4; sphingomyelin (Sph), 28.4 and lysolecithin (LL), 1.7. The fatty acid composition of each phospholipid was determined by GLC. The average number of double bonds per fatty acid in the isolated phospholipids was found to be as follows: PI, 1.5; PE, 1.9; EP, 3.6; PS, 2.1; L, 1.0; CP, 2.0; Sph, 0.2 and LL, 0.5. The positional distribution of fatty acids in both L and PE was ascertained by selective enzymatic hydrolysis with phospholipase A. Saturated fatty acids of L were esterified predominantly in the α′-position, whereas in PE only 63.9 mole per cent of the saturated fatty acids were found in this position. Presented in part at the AOCS Meeting in Los Angeles, April 1966. Dept. of Health, Education and Welfare, USPHS.     

Fatty acids of bovine milk glycolipids and phospholipids and their specific distribution in the diacylglycerophospholipids

Milk lipids were fractionated by silicic acid column chromatography and preparative thinlayer chromatography (TLC). Ceramide monohexoside (CMH), ceramide dihexoside (CDH), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), phosphatidyl serine (PS), and sphingomyelin (Sph) were isolated, and the purity of each was checked by infrared spectroscopy and TLC. The diacylphospholipids were hydrolyzed with phospholipase A and the products separated by TLC. Fatty acid methyl esters were prepared from the various fractions and analyzed by gas chromatography. The glycolipids, CMH and CDH, and Sph contained large amounts of long-chain saturated fatty acids, especially C_22:0, C_23:0, and C_24:0, PE, PS, and PC contained C₁₀-C₂₂ normal and branched-chain saturated fatty acids, and C₁₅-C₂₀ unsaturated fatty acids (mainly monoenes). The distributions of saturated acids between the α′- and β-positions were respectively: PE, 46 and 11%; PS, 65 and 19%; and PC, 72 and 53%. PC was exceptional in that there was 10.8% myristic acid in the β-position and only 5.6% in the α′-position. PE and PS were similar in composition except that in PE oleic acid was evenly distributed, and in PS was largely in the β-position. In general, PC was much more saturated than PE or PS, and there was no overall pattern governing the specific distribution of the fatty acids in the three diacylphospholipids. Comparison with PC from other bovine tissues and from egg lecithin showed that fatty acids are located much less specifically in milk phospholipids than in PC from other sources. Presented at the AOCS Meeting, Houston, Texas, April, 1965.     

The effect of diet on the phospholipid composition of the red blood cells of man

Short term (16 day) controlled fat (formula type diet) feeding to 10 healthy adult males led to no detectable change in the total amt or the relative proportions of the individual phospholipids of the red blood cells, although limited changes did occur in the fatty acids of certain of the phospholipids. The total phospholipid content of the red blood cells was 315±10 mg/100 ml (average of 20 samples). Lecithin accounted for 34% of the total, with sphingomyelin, phosphatidyl ethanolamine and phosphatidyl serine representing 25, 25 and 16%, respectively. Approx 36% of the phosphatidyl ethanolamine, 4% of the phosphatidyl serine and 6% of the lecithin was present in the plasmalogen form. Each phospholipid class was found to have a distinctive fatty acid spectrum. The M ratio of saturated to unsaturated fatty acids in all three phosphoglycerides was nearly 1:1. Behenic, lignoceric and nervonic acids made up almost half of the sphingomyelin fatty acids, and the M ratio of saturated to unsaturated fatty acids in this lipid was 3:1. When compared with red cells from subjects consuming a diet with a high butter fat content, red cells from subjects on a diet rich in corn oil were found to contain higher levels of linoleic acid in the lecithin and phosphatidyl serine fractions, and lower levels of oleic acid in the lecithin fraction. No changes were observed in the fatty acids of the phosphatidyl ethanolamine and sphingomyelin fractions. It is probable that these alterations represent the result of highly specific exchanges with plasma fatty acids, and it is suggested that three levels of specificity are involved: class of phospholipid, type of fatty acid, and specific fatty acid.     

Quantitative analysis of brain and spinach leaf lipids employing silicic acid column chromatography and acetone for elution of glycolipids

Quantitative elution of acidic and neutral glycolipids of brain and spinach leaves from silicic acid columns with acetone was demonstrated. Cerebrosides and sulfatides of brain and sulfolipid and glycosyl diglycerides of spinach leaves were eluted quantitatively with acetone while prospholipids remained on the column. The observations provide the basis for an analytical procedure employing column and quantitative thin-layer chromatography (TLC). Sephadex column chromatography is utilized for separation of lipids from nonlipids; silicic acid column chromatography for separation into neutral lipid, glycolipid and phospholipid fractions; and quantitative TLC for analysis of lipid classes of each column fraction.     

Analysis of Phospholipids in Rat Brain Using Liquid Chromatography–Mass Spectrometry

The brain is a lipid-rich organ containing complex polar lipids including phospholipids (PLs) and sphingolipids. These lipids are involved in the structure and function of cell membranes in the brain. We developed a fast and efficient liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to quantify five different classes of PLs [Choline glycerophospholipid (consists of phosphatidyl choline and plasmenyl choline in these samples), ethanolamine glycerophospholipid (consist of phosphatidyl ethanolamine and plasmenyl ethanolamine in these samples), phosphatidyl serine, phosphatidyl inositol, and sphingomyelin] in the brain tissues of 80-day-old Wistar rats. The PLs were extracted from rat brain using chloroform/methanol/water. After separation using a hydrophilic high performance liquid chromatography column, PL-class-specific fragmentation (head group identification) with a tandem mass spectrometer in positive ion mode was utilized to measure changes in the relative concentration of the five PL classes. The advantage of this approach was its improved specificity over previously reported LC–MS methods. The method had good repeatability (coefficient of variation 3–9%, excluding phosphatidyl inositol) and recovery (92–103%) and compared well with more laborious traditional methods.