Toolbox for modification of the lecithin headgroup

In an effort to produce structurally divergent lecithins for testing of their functional properties and use in food products, several tools have been developed to carry out modifications in the polar head group distribution of the native phospholipids in soybean lecithin. These tools include physical, chemical and enzymatic techniques. Using a combination of acetone de‐oiling, ethanol fractionation, N‐acetylation and enzymatic hydrolysis and transphosphatidylation with a phospholipase D from Streptomyces sp., a set of lecithins with modified head group distributions were produced. The kinetics of the enzymatic head group hydrolysis and transphosphatidylation was studied in detail. Reaction rates and selectivity (transphosphatidylation / hydrolysis) were affected by both lecithin concentration and donor alcohol concentration. Hydrolysis, forming phosphatidic acid, was strongly dependent on both concentrations, whilst transphosphatidylation, forming phosphatidyl glycerol (or phosphatidyl ethanolamine or phosphatidyl serine), was only influenced by the donor alcohol. This resulted in a reduction in selectivity at high initial lecithin concentrations and suggested the use of a reactant feeding strategy. Enrichment of the phosphatidyl choline content of native soybean lecithin was achieved by ethanol fractionation and phosphatidyl inositol enrichment was by N‐acetylation with acetic anhydride followed by de‐oiling. The application of these tools, together with others designed to modify the fatty acid composition of phospholipids, was used to produce 10‐100 g quantities of divergent lecithins and can be routinely used at lab‐scale.     

Analysis of soybean lecithin by thin layer and analytical liquid chromatography

The application of thin layer and analytical liquid chromatography to the analysis of two samples of commercial soybean lecithins is described. A combination of column chromatography and quantitative thin layer chromatography showed that these products consisted of ca. 82% mixture of the major phospholipids of soybeans, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol. The remainder of these products contained essentially the entire spectrum of lipid classes found in soybean oil-some 24 known and unknown glycolipids and phospholipids, in addition to the neutral lipids. Applications of analytical liquid chromatography to these lecithins gave a composition profile of the lipid classes comparable to two-dimensional thin layer chromatography. The potential of this method for the complete analysis of complex lipids, such as soybean lecithins, is indicated.     

Phospholipids of pork muscle tissues

The lipids extracted from four locations on a hog carcass were fractionated into nonphospholipids, phosphatidyl ethanolamine, phosphatidyl serine, lecithins, and sphingomyelins. The identities of phospholipid fractions were established and their quantitative contents were determined by three methods: gravimetrically, from analysis of the phosphorus content, and from standard curves of infrared spectra. Variations in content of a phospholipid type were noted in the carcass locations studied. Lecithin and phosphatidyl ethanolamine were found in greatest quantity. These represented ca. 61% and 31%, respectively, of the total phospholipids. The remainder was distributed between phosphatidyl serine and sphingomyelin at 5% and 3%, respectively. The phospholipid content of hog meat was found to be slightly over 0.5%. Journal Article No. 3077, Michigan Agricultural Experiment Station. Supported by research grant No. RG-8801, National Institutes of Health, U. S. Public Health Service, Department of Health, Education, and Welfare. Presented at the AOCS meeting in Toronto, Canada, 1962.     

Emulsifying Properties of Different Modified Sunflower Lecithins

Lecithins are a mixture of acetone-insoluble phospholipids and other minor substances (triglycerides, carbohydrates, etc.). The most commonly processes used for lecithin modification are: fractionation by deoiling to separate oil from phospholipids, fractionation with solvents to produce fractions enriched in specific phospholipids, and introduction of enzymatic and chemical changes in phospholipid molecules. The aim of this work was to evaluate the emulsifying properties of different modified sunflower lecithins in oil-in-water (O/W) emulsions. In this study, five modified sunflower lecithins were assessed, which were obtained by deoiling (deoiled lecithin), fractionation with absolute ethanol (PC and PI enriched fractions), and enzymatic hydrolysis with phospholipase A₂ from pancreatic porcine and microbial sources (hydrolyzed lecithins). Modified lecithins were applied as an emulsifying agent in O/W emulsions (30:70 wt/wt), ranging 0.1–2.0% (wt/wt). Stability of different emulsions was evaluated through the evolution of backscattering profiles (%BS), particle size distribution, and mean particle diameters (D [3, 4], D [3, 2]). PC enriched fraction and both hydrolyzed lecithins presented the best emulsifying properties against the main destabilization processes (creaming and coalescence) for the analyzed emulsions. These modified lecithins represent a good alternative for the production of new bioactive agents.     

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.     

Phospholipids of the pulmonate land snailCepaea nemoralis (L.)

The phospholipids of the snailCepaea nemoralis, comprising the major lipid fraction (65%) in this terrestrial pulmonate, were investigated by thin-layer and column chromatography. Detailed gas chromatographic analyses of liberated fatty acid fractions and amino acid analyses of the water soluble moieties of isolated phospholipid classes were carried out. Phosphatidyl choline (47%) and phosphatidyl ethanolamine (21%) were found to be the predominant phospholipid classes, while phosphatidyl serine (8%), phosphatidyl inositol (6%), diphosphatidyl glycerol (3%), ceramide amino-ethylphosphonate (7%), lysophosphatidyl choline (1%), and phosphatidic acid (1%) were present in lesser amounts. In the phosphatidyl ethanolamine and phosphatidyl serine fractions, minor quantities of plasmalogen analogues were detected. Fatty acid profiles of the various phospholipid classes appeared to be strikingly diverse, e.g. a characteristic component, such as linoleic (18∶2ω6) acid, ranging from 3–54%. In vivo radioisotope studies using 1-¹⁴C-acetate demonstrated the high biosynthetic rate of all phospholipid classes and their respective fatty acid fractions. Results are discussed in relation to data on the phospholipids from other invertebrate species.     

Lipids ofKlebsiella pneumoniae: The presence of phosphatidyl choline in succinate-grown cells

The carbon and energy source for aerobically grown cultures ofKlebsiella penumoniae profoundly influenced the total lipid content and phosphatide composition. Glucose-grown cells contained 13% lipid, 56% of which was phospholipids. Succinate-grow cells contained 8% lipid, 66% of which was phospholipids. The predominant phosphatides of glucose-grown cells were phosphatidyl ethanolamine, 82%; phosphatidyl glycerol, 4.5%; phosphatidic acid, 5%; cardiolipin, 6.5%; phosphatidyl serine; and trace amounts of unidentified phosphatides. Phosphatides of succinate-grown cells were phosphatidyl ethanolamine, 38%; diphosphatidyl glycerol, 14%; phosphatidyl glycerol, 13%; phosphatidyl choline, 14.5%; phosphatidyl serine, 6%; phosphatidic acid, 4%; and 10% unknown lipids. No trace of phosphatidyl choline was found in glucose-grown cells. Paper 75-11-170 of the Kentucky Agricultural Experiment Station.     

Toolbox for exchanging constituent fatty acids in lecithins

Since the compositional variety of phospholipids in native lecithins is limited, and structurally diverse synthetic phospholipids are expensive, the aim of this work was to develop a lecithin modification toolbox capable of modifying the fatty acid composition of native lecithins at a convenient lab‐scale of 10‐100 g. Starting with the native phospholipids of soybean lecithin, two types of fatty acid modification were carried out using either phospholipase A2 from porcine pancreas or a lipase from Rhizopus oryzae. The former was immobilised onto celite and used to selectively hydrolyse the sn‐2 positioned fatty acid and the latter, commercially available in an immobilised form, was used to transesterify novel fatty acids onto the sn‐1 position. The degree of phospholipid hydrolysis could be controlled between 5 and 95% by varying the contact time with the biocatalyst. A key parameter was the water concentration. By avoiding the presence of a bulk phase of water, emulsion formation was prevented and so simple product recovery was possible. However, sufficient water was required in order to maintain the water activity above 0.2, and because phospholipids increased the polarity of the solvent (hexane), it was necessary to add water in proportion to the lecithin concentration. During transesterification with methyllaurate, up to 43% lauric acid could be incorporated and although the recovery of fully acylated phospholipids was only 28%, due to the formation of hydrolysis products, they were isolated using solvent partitioning in hexane/isopropanol/water. The temperature was important in determining the relative rates of hydrolysis and transesterification and a lower temperature (40 °C) was favourable as the rate of hydrolysis was lowered without affecting the rate of transesterification.     

Biosynthesis of phospholipids in subcellular particles from cultured cells of human tissue

A time study of the incorporation of³²P_i into the phospholipids of HeLa, KB, human heart, and liver tissue-culture cell lines has been carried out. The incorporation of³²P_i at various time-intervals into the phospholipids of nuclei, mitochondria, and microsomes of HeLa and KB cells was investigated. The labeling of the isotope into the phospholipids was divided into three groups. The first had two components: phosphatidyl inositol and polyglycerol phosphatides, which showed the greatest incorporation of the isotope as demonstrated in the specific activity values and the percentage of total radioactivity after 15 to 30 minutes of incubation. A second group was composed of the major phospholipids of all tissue-culture cell lines studied, phosphatidyl choline, and phosphatidyl ethanolamine. At first, there was a delayed labeling of these phospholipids; however, after one hour of incubation, a rapid increase was shown in the incorporation of³²P_i. A third group of lipids containing sphingomyelin and phosphatidyl serine demonstrated low specific activity values. The phospholipids of the subcellular fractions, nuclei, mitochondria, and microsomes, had a high degree of incorporation of the isotope into the individual phospholipids and probably represented an active process in the membranes of these cellular units or a renewal of the biological membrane structures. Part of a thesis submitted to the Graduate School of the University of North Dakota in partial fulfillment for the degree of Doctor of Philosophy.     

Fatty acid composition of milk phospholipids. III. Camel,ass, and pig milks

Phospholipids were isolated from camel, ass, and pig milks, and their fatty acid compositions were determined by gasliquid chromatography. The specific distributions of fatty acids in phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) were determined. The results are compared with previous results for bovine, sheep, Indian buffalo, and human milks. The milk phospholipids which were studied can be grouped, on the basis of their fatty acid compositions, into those from ruminant herbivores, nonruminant herbivores, and nonherbivores. The phospholipids of camel milk however have features typical of all groups as well as 15% plasmalogen in the PE fraction. For Parts I and II, see References 1 and 2.     

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.     

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.     

Update on vegetable lecithin and phospholipid technologies

This paper reviews the production technologies for sourcing lecithins from the oil‐bearing seeds soybean, rapeseed and sunflower kernel. The phospholipid composition is measured by newly developed HPLC‐LSD and ³¹P‐NMR methods. The phospholipid compositions of the three types of lecithin show small differences, while the fatty acid composition is largely equivalent to the oil source. Regulatory specifications (FAO/WHO, EU, FCC) and DGF and AOCS analytical methods for product quality are compiled. Phospholipid modifications by enzymatic hydrolysis, solvent fractionation, acetylating and hydroxylation processes result in lecithins with specific enhanced hydrophilicity and oil‐in‐water emulsifying properties. New available phospholipase and lipase enzymes represent opportunities for the esterification of phospholipids with special omega fatty acids and serine groups. Application characteristics are given for use in yellow fat spreads, baked goods, chocolate, agglomerated instant powders, liposome encapsulation, animal feed, food supplements and pharmaceutics.     

Antioxidant effect of soy lecithins on vegetable oil stability and their synergism with tocopherols

The antioxidant effect of lecithins was tested on several oils and fats varying in FA composition and tocopherol content. Standard lecithins, when added at a level of 1% w/w, exhibited a good protective effect against oxidation. This effect was observed to depend on the phospholipid content of the tested lecithins and the FA composition of the tested oils. Better results were obtained with lecithin samples containing high proportions of PC and PE. Indeed, the main antioxidant mechanism of lecithins was due to a synergistic effect between amino-alcohol phospholipids and γ- and δ-tocopherols. No synergism was observed with α-tocopherols, especially when the tested oil was rich in linoleic acid. Therefore, the antioxidant protection of lecithins was not effective for sunflower oil. Finally, the use of fractionated or enriched lecithins was not clearly advantageous compared to standard oil lecithins.     

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.     

Studies on tamarind kernel oil II1L : analysis of phospholipids

Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phospha-tidic acid, and lyso derivatives of phosphotidyl choline and phosphatidyl ethanolamine were identi-fied from phospholipids of Tamarind kernel oil. Phosphatidyl choline and phosphatidyl ethanolamine were isolated and their fatty acid composition was determined by GLC. Reference 2 is considered as paper I of the series. Presented at Annual Convention of Chemists, Dec. 1977, at Jaipur, India.     

Studies on the lipids of sheep red blood cells. II. The incorporation of phosphorus into phospholipids of HK and LK cells

The incorporation of inorganic phosphate (as NaH₂PO₄) into the phospholipids of sheep red blood cells was studied in vitro in blood samples from five highpotassium (HK) and five low-potassium (LK) sheep. The erythrocytes from HK sheep incorporated more activity in 4 hr than those from the LK sheep. However no activity was incorporated into the major phospholipids of the cells (phosphatidyl ethanolamine, phosphatidyl serine, and sphingomyelin) of either group. The phosphatidic acid fraction was labeled in both groups and to a significantly greater extent in the HK samples. However the highest activity in the phospholipid of sheep red-cells was located in three unknown compounds not previously detected. Their specific activities were the same in the HK and the LK samples although they were present in slightly larger amounts in the HK samples. In general, incorporation was at a rather low level, and from stoichiometric considerations it was concluded that the metabolism in the red-cell phospholipids could not be directly involved in the active transport of ions across the cell membrane. This work also confirmed a previous report that no quantitative differences exist among the major phospholipid classes in the two types of cells.     

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.     

Effect of lecithins partly replacing rumen‐protected fat on fatty acid digestion and composition of cow milk

The effects on fatty acid digestibility and milk fat composition of calcium soaps of palm oil fatty acids and of a 25% replacement of the Ca soaps by four different lecithins (raw, deoiled and deoiled/partially hydrolysed soy lecithin, raw canola lecithin) and soybean oil were investigated in six lactating cows each. The complete diets contained the lipid supplements at proportions of 30 g fatty acids/kg dry matter. Partial replacement of Ca soaps by soy or canola lecithins and soybean oil had small but significant effects on fatty acid digestion and utilisation, as well as the fatty acid profile in milk. Relative to Ca soaps alone, C 16:0 digestibility was slightly higher with lecithins, and percentage of conjugated linoleic acid and trans C 18:1 in milk fat increased while proportion of C 16:0 decreased. Deoiling of lecithins slightly reduced the effects on C 16:0 digestibility and excretion with milk. The influence of lecithin processing was higher than the differences between raw soy and raw canola lecithin. Nevertheless, most of the few effects observed may be related to the fatty acids supplied with the lecithins but, regarding C 18:1 trans‐11 and odd chain fatty acids, there is some evidence that lecithins impair rumen microbial activity less than soybean oil.