Chemical composition of subcellular particles from cultured cells of human tissue

Chemical composition of subcellular components of HeLa, KB, human heart and liver tissue-culture cell lines have been studied. The concentration of RNA, protein and phospholipid (μg/μg of DNA) of total subcellular particles was similar for all four cell lines studied. The greatest RNA concentration and lowest protein concentration is found in the microsomes as compared to the other subcellular fractions of HeLa and KB cells. The lipid P/Protein N ratio of mitochondria was greater than the other subcellular fractions from tissue-culture cell lines studied. Phosphatidyl choline and phosphatidyl ethanolamine are the major phospholipids with the former more predominant in all of the subcellular fractions of tissue-culture cells studied. Phosphatidyl inositol, phosphatidyl serine, sphingomyelin and polyglycerol phosphatide were shown to be present. Phosphatidyl choline composition (per cent of total lipid-P) is greatest in the microsomes when compared with the other subcellular fractions obtained from all of the cell lines studied except the nuclear fraction of human liver cells. Correspondingly, the mitochondrial fraction for all of the tissue culture cell lines contains the greatest composition of phosphatidyl ethanolamine except for the human liver and heart cells. The mitochondrial fraction contains the lowest amount of phosphatidyl inositol. Polyglycerol phosphatide is mainly present in the mitochondrial fraction of the tissue-culture cells. 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.     

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.     

Lipid composition ofNeurospora crassa

The lipids ofNeurospora crassa, isolated in pure form from freeze-dried mycelium, were found to contain squalene, sterol esters, triglycerides, free fatty acids, geranylgeraniol, free sterols, carotenoids, cardiolipin, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, and phosphatidic acid. The above compounds were isolated in pure form by column and thin layer chromatography and were characterized by infrared spectroscopy and chromatographic mobilities. Fatty acid moieties were characterized by gas liquid chromatographic retention times of their methyl esters relative to those of authentic standards. The fatty acid composition of the triglycerides was found to be similar to that of phosphatidic acid, cardiolipin, and lecithin.     

Lipid composition of 30 species of yeast

The detailed composition of cellular lipid of more than 23 species of yeast has been determined quantitatively by thinchrography on quartz rods, a method previously used for estimating cellular lipids of seven species of yeast. That data was fortified by neutral and phospholipid quantitations on 30 species of yeast cells. Most of the test organisms contained 7–15% total lipid and 3–6% total phospholipid per dry cell weight, except for the extremely high accumulation of triglycerides in two species ofLipomyces. Qualitatively, 30 species of yeast cells contained similar neutral lipid constituents (triglyceride, sterol ester, free fatty acid, and free sterol) and polar lipid components (phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, cardiolipin, and ceramide monohexoside) without minor constituents. Based on the quantitative composition of neutral lipids, the 30 species of yeast were divided into two groups, the triglyceride predominant group and the sterol derivative group. These groupings were fairly well overlapped from the standpoint of the distribution characteristics of fatty acid. The relative polar lipid compositions also grossly resembled each other. Only one exception of polar lipid composition in yeast cells was found inRhodotorula rubra species which contained phosphatidyl ethanolamine as the most abundant phospholipid. Fatty acid distribution patterns in yeast cells consistently coincided with other reports concerning fatty acid composition of yeast cells. Correlation of lipid composition and classification of yeasts are suggested and discussed. A part of this investigation has been reported at the 14th conference of the Japan Oil Chemists' Society, Nagoya, Japan, October 1975.     

Lipid composition of normal and hypertrophic bovine thyroids

The phospholipid content of bovine thyroid tissue amounts to 70% of total lipid. Triglycerides and cholesterol are the main neutral lipids. Only trace amounts of free fatty acid and esterified cholesterol are found, while two not yet identified components also are present. The distribution of lipid phosphorus in the different phospholipid classes is as follows: phosphatidyl choline, 43.0%; phosphatidyl ethanolamine, 25.2%; phosphatidyl serine, 5.6%; phosphatidyl inositol, 6.5%; sphingomyelin, 14.0%; cardiolipin, 2.8%; lysophosphatidyl choline, <1%; and phosphatidic acid, <1%. The phosphatidyl ethanolamines are rich in plasmalogens. The fatty acid patterns in the different lipid classes are reported. The essential differences between normal and hypertrophic bovine thyroid tissue are higher water content and lower triglyceride and sphingomyelin values for hypertrophic tissue.     

Lipid composition and endogenous respiration of pig heart mitochondria

Lipid composition and endogenous respiration of pig heart mitochondria were studied in parallel, since the level of endogenous respiration affects the oxidation of added substrates and therefore the regulation of oxidative phosphorylation; mitochondrial lipids can interfere either as substrates or as partner in the energy conservation mechanism. O₂ uptake kinetics were measured in presence of different additives: ATP, ADP, NAD⁺ and hexokinase + glucose. The lipid composition of pig heart mitochondria was determined by chromatographic and spectrophotometric methods. Total lipids were 90% phospholipids; the main phosphatides were cardiolipin, phosphatidyl choline and phosphatidyl ethanolamine; the two latter were rich in plasmalogens. The main nonpolar lipids were triglycerides and free fatty acids. The fatty acid composition of total lipids, phospholipids, free fatty acids and triglycerides was determined by gas liquid chromatography. Mitochondrial lipids were characterized by a high content of unsaturation. Part of this work is included in “Thèse de Doctorat de Spècialitè en Biochimie” de J. Comte, Lyon, June 26, 1970.     

Distribution of cholesteryl esters and other lipid in subcellular fractions of the adrenal gland of the pig

Total lipids from whole pig adrenal glands as well as from their mitochondria, microsomes, liposomes, and cell sap were extracted and fractionated first into neutral lipids and phospholipids. The highest percentage of neutral lipids was found in the cell sap, and the lowest in the microsomal fraction. Neutral lipids were subfractionated into cholesteryl esters, free cholesterol, triglycerides, and free fatty acids. Cholesteryl esters were distributed throughout the liposomes. Free fatty acids represented a substantial part of cell sap lipids, but were present also in the mitochondria, microsomes, and liposomes. Fatty acids of all fractions were analyzed by gas liquid chromatography. Free fatty acids and cholesteryl ester fatty acids from all cellular fractions were similar in composition and were characterized by considerable quantities of linoleic and arachidonic acid. Triglycerides were characterized by an increased percentage of palmitic and a low content of arachidonic acid. Phosphatidyl choline, phosphatidyl ethanolamine, diphosphatidyl glycerol, and sphingomyelin plus phosphatidyl inositol were isolated from the lipids by preparative thin layer chromatography, and their fatty acids analyzed by gas liquid chromatography. Phosphatidyl choline and phosphatidyl ethanolamine from mitochondria, microsomes, and cell sap were very similar in respect of their fatty acid composition. Sphingomyelin plus phosphatidyl inositol was characterized by a high content of C22:2omega6. Diphosphatidyl glycerol was present in mitochondria and in the cell sap.     

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.     

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.     

Lipid composition of beef brain,beef liver,and the sea anemone: Two approaches to quantitative fractionation of complex lipid mixtures

Two new schemes for fractionation of complex lipid mixtures are presented. Their use for the study of lipids of beef brain, beef liver, and the sea anemone are described. Apparatus and techniques for working in an inert atmosphere, evaporation of solutions in the cold under nitrogen, use of infrared spectroscopy for examination of lipids and their hydrolysis products, preparation and clution of diethylaminoethyl (DEAE) cellulose and silicic acid-silicate columns and general column combinations that can be used to fractionate complex lipid mixtures are considered in detail. The first scheme, employing DEAE cellulose columns followed by thin layer and paper chromatographic examination of the fractions, was applied to liver lipids. The many components, some of them new lipids not previously detected, are clearly seen with this technique but are not seen when paper or thin layer chromatography alone or silicic acid chromatography are used. The second scheme employing DEAE for initial fractionation, followed by complete separation on silicic acid and silicic acid-silicate columns, was applied to lipids of the sea anemone and beef brain. Typical lecithin and phosphatidyl ethanolamine were isolated, but sphingomyelin was not found. A new sphingolipid, ceramide aminoethylphosphonate, with a free amino group and a direct carbon to phosphorus bond was isolated and characterized. The methods used for quantitative isolation, the infrared spectra, and the amounts of cholesterol, ceramide, cerebroside, galactosylglyceride, sulfatide, sphingomyelin, lecithin, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, triphosphoinositide, phosphatidic acid, cardiolipin, and ganglioside of beef brain are presented. Finally, the types of lipid-nonlipid interactions disclosed by column chromatography and their potential application to biological problems are discussed.     

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.     

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.     

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.     

The occurrence and biosynthesis of gamma-linolenic acid in a blue-green alga,Spirulina platensis

The acyl-lipid and fatty acid composition of six blue-green algae, namely,Spirulina platensis, Myxosarcina chroococcoides, Chlorogloea fritschii, Anabaena cylindrica, Anabaena flos-aquae, and Mastigocladus laminosus is reported. All contain major proportions of mono-and digalactosyl diglyceride, sulfoquinovosyl diglyceride, and phosphatidyl glycerol, but none possess lecithin, phophatidyl ethanolamine, or phosphatidyl inositol. Trans-3-hexadecenoic acid was absent from all extracts. The analyses provide further evidence that there is no general chemical or physical requirement for any specific fatty acid in photosynthesis. S. platensis is unique among photoautotrophic organisms so far studied, containing major quantities of γ-linolenic acid (6,9,12-octadecatrienoic acid). This acid is synthesized by the alga by direct desaturation of linoleic acid and is primarily located in the mono- and digalactosyl diglyceride fractions. The possible phylogenetic relationship betweenS. platensis and other plant forms is discussed.     

Studies on the lipids of sheep red blood cells. I. Lipid composition in low and high potassium red cells

The lipid composition of whole red blood cells was investigated in five sheep with red cells containing a low concentration of potassium (LK) and in five sheep with red cells containing a high concentration of potassium (HK). No apparent differences within the limit of error of the experiment were detected in the lipid class composition between the HK and LK red cells. Cholesterol, the only nonpolar lipid detected in the tissue, was present in oneto-one molar ratio to the total phospholipids. Phosphatidyl ethanolamine and sphingomyelin accounted for 85% of the total phospholipids; phosphatidyl serine, phosphatidyl inositol, phosphatidic acid, and lysolecithin were present in lesser amounts. No lecithin was detected in any of the animals in this investigation. Plasmalogen compounds were found only in the ethanolamine lipids. The molar ratio of choline to noncholine phospholipids was also approximately one to one. It was concluded that the major lipid class distribution in the two types of red cells cannot be directly responsible for the differences observed in the cation concentrations in these cells in the two species of sheep.     

Phospholipid Composition of <Emphasis Type="Italic">Jatropha curcus</Emphasis> Seed Lipids

Jatropha curcus L. oil has emerged as one of the most important raw materials for biodiesel production. However, no detailed study has been reported on characterizing the lipid constituents of jatropha oil. The present study revealed that the total oil content of jatropha seeds was 32% with a composition of 97.6% neutral lipids, 0.95% glycolipids and 1.45% phospholipids. The fatty acid composition of total lipids, neutral lipids, phospholipids and glycolipids was also determined and found to contain oleic acid (18:1) and linoleic acids (18:2) as major fatty acids. The phospholipids fraction was further characterized and quantified and found to contain phosphatidyl choline (PC) 60.5%, phosphatidyl inositol (PI) 24% and phosphatidyl ethanolamine (PE) 15.5%. The fatty acid composition and the positional distribution of the fatty acids of individual phospholipids were also reported.     

Liver lipids during development

The fatty acid composition of the major liver microsomal phospholipids has been studied during pre- and postnatal development of the rabbit. The fatty acid composition of the total lipids, phosphatidyl choline, and phosphatidyl ethanolamine from animals −6, −3, 0, +3, +6, +9, +16, and +112 days of age was determined. Fatty acid composition is similar in phosphatidyl choline and phosphatidyl ethanolamine for oleic acid at +3, +6, +9, and +16 day old animals; palmitoleic acid at +9 day old animals and linoleic acid at −6, −3, and 0 day old animals. Palmitoleic acid demonstrated a uniform decrease during early development in the total lipids and in both phosphatidyl choline and phosphatidyl ethanolamine; however, in the 112 day animal, the amount was just slightly lower than that observed for the earliest prenatal animal studied. Oleic acid decreased considerably during early postnatal development in the total lipids, phosphatidyl choline and phosphatidyl ethanolamine, but an increase in the 112 day animal was observed. Linoleic acid fluctuated considerably throughout postnatal development in the total lipids as well as in the two major phosphatides. Lecithin biosynthesis has been studied by two pathways during development of rabbit liver from −6 days to +110 days. The two pathways of lecithin biosynthesis were evaluated by assaying the activities of the liver enzymes choline phosphotransferase and phosphatidylmethyltransferase at different time intervals during development. The greater enzymatic activity was observed in the cholinephosphotransferase during development.     

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.     

Reaction of anti-phosphatidyl inositol antisera with neural membranes

Ca. 15% of the phosphatidyl inositol in myelin and microsomal membranes from rat brain was detectable by antiphosphatidyl inositol antibody. Antibody-detectable phosphatidyl inositol in myelin and microsomes readily increased when the membranes were incubated at 45 C with the antiserum. Synaptic membranes also had only a limited capacity to adsorb antibody. Quantitative binding studies with synaptic membranes and mitochondria were limited, because these membranes contain cardiolipin, which cross reacts with phosphatidyl inositol antisera. Moreover, highly purified synaptic and mitochondrial membranes contain appreciable amounts of other membrane fractions.     

Study of free and bound lipids ofBrassica campestris,var yellow sarson

Mature seeds ofBrassica campestris var. yellow sarson were extracted with hexane to yield free lipid. The residue then was extracted with chloroform-methanol to release bound lipid. Free and bound lipids were separated into polar and nonpolar fractions chromatographically. The nonpolar fraction of both free and bound lipid consisted mainly of triglycerides with small amounts of steryl esters, free sterols, mono- and di-glycerides, and free fatty acids. The principal components of polar bound lipid were phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and steryl glycoside. In the free polar lipid, there was more phosphatidyl inositol and less phosphatidyl choline and phosphatidyl ethanolamine. Erucic acid content was much greater in the nonpolar fractions and in the polar free lipid than in the polar bound lipid.