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.     

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.     

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.     

Phospholipid composition of the boll weevil,Anthonomus grandis boheman

When phospholipids of newly-emerged adults of the boll weevil,Anthonomus grandis Boheman, were studied in detail, phosphatidyl choline and phosphatidyl ethanolamine were found to be the major phospholipids; sphingomyelin and cardiolipin were present in smaller amounts, and four other minor components were identified. Fatty acid analyses performed on the intact phospholipids and on the enzyme degradation products of phosphatidyl choline and phosphatidyl ethanolamine demonstrated that oleic and linoleic acids were the major fatty acids present in the glycerophosphatides; the sphingomyelin contained fatty acids in the range of 20–22 carbons.     

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.     

Molecular species of phosphatidyl ethanolamine from egg yolk

Phosphatidyl ethanolamine was isolated from total egg yolk lipids by preparative thin layer chromatography (TLC). The purified phosphatide contained 3% of the alkoxy derivative. It was degraded to diglycerides in the presence of purified sphingomyelin by phospholipase C fromClostridium welchii. The diglycerides were acetylated and resolved on the basis of unsaturation by argentation TLC. The fatty acid composition of the original phosphatidyl ethanolamine and the derived acetates was determined by gas chromatography, as was the molecular weight distribution of the diglyceride acetates. The placement of the fatty acids in the parent phosphatide was deduced by hydrolysis with phospholipase A fromCrotalus atrox, and in the acetates with pancreatic lipase. Some 33 major species of phosphatidyl ethanolamine were identified and compared to those for egg yolk lecithins. Presented in part at the Canadian Federation of Biological Societies Meeting, Kingston, June 1968.     

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.     

Studies on liver phosphatidyl cholines: II. Effect of sex,age and species differences on phosphatidyl cholines from liver mitochondria and microsomes

Liver mitochondrial and microsomal phosphatidyl cholines differing in the degree of unsaturation of their fatty acids have been separated into four fractions by silver ion silica gel TLC. The levels of the four phosphatidyl choline fractions were determined for male and female rats and mice, fetal and young rabbits, and female hamsters and guinea pigs. The sum of phosphatidyl choline fractions 1, 2, and 3 of mitochondria and microsomes was greater in the female rat than in the male rat with the difference being a reflection of a higher level of fraction 3 which contains arachidonic acid. The female rat has greater concentration of phosphatidyl choline fractions 1 and 3 of mitochondria. Similar results were seen in mouse liver microsomes but not in mitochondria. The levels of the individual four fractions varied from species to species. No change occurred in the levels of the phosphatidyl choline fractions of fetal (−9 and −3 days) rabbits, but an increase was seen in the level of fraction 4 between day 3 and day 35 in both the mitochondria and microsomal fractions of liver. The concentration of mitochondrial and microsomal protein, total phospholipid and total lecithin phosphorus were determined in rat, mouse, hamster and guinea pig. The total phospholipid phosphorus/protein (μg/mg) of microsomes was greater in all species than that observed in mitochondria. Liver microsomes contain 45–50% of total phospholipid phosphorus as lecithin whereas mitochondria contains 32–37%. The fatty acid patterns of mitochondria and microsomal phosphatidyl cholines were determined and the ratio of palmitate to stearate was greater than two for mice and hamsters and approximately 0.5 for rat and guinea pigs.     

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.     

On the membrane phospholipids and their acyl group profiles of adrenal gland

The phospholipid composition and their acyl group profiles from subcellular fractions of guinea pig adrenal gland and the same fractions from the cortex and medulla of the bovine gland were compared. The phospholipids of guinea pig adrenal were enriched in diacyl-glycerophosphocholines (GPC) which comprised over 50% of the total phospholipids, but the proportions of ethanolamine and choline plasmalogens, sphingomyelin and diacyl-glycerophosphoserine (GPS) were lower in guinea pig adrenals as compared to the bovine adrenals. In the bovine adrenal, sphingomyelin and diacyl-GPS were enriched in the medulla, whereas diacyl-glycerophosphoinositol (GPI) were enriched in the cortex. Although lysolecithin was present (up to 4.5%) in the bovine adrenal, only trace amounts of this lipid were detected in the guinea pig adrenal. Characteristic acyl group profiles were found associated with each type of the phosphoglycerides in adrenal membranes. However, acyl group profiles of the phosphoglycerides were not greatly different either between the bovine and guinea pig adrenal or with respect to the type of subcellular membranes isolated. Diacyl-GPC were enriched in 16∶0 and 18∶1, but also contained considerable amounts of 18∶0, 18∶2 and 20∶4. Diacyl-GPE were enriched in 18∶0, 18∶1 and 20∶4, while diacyl-GPI, diacyl-GPS, as well as alkenylacyl-GPE, were enriched in 20∶4. The lysolecithin from bovine adrenal membranes contained mainly 16∶0, 18∶0 and 18∶1 with only trace amounts of the polyunsaturated fatty acids. Other polyunsaturated fatty acids, such as 22∶4 and 22∶6, are apparently not prominent in the phosphoglycerides from either the bovine or the guinea pig adrenal gland.     

The renal phospholipid composition of choline-deficient rats

The base composition of the phospholipids involved in the N-methylation pathway for the biosynthesis of phosphatidyl choline was determined in normal and severely hemorrhagic rat kidneys. There was a decrease in the proportion of phosphatidyl choline and phosphatidyl ethanolamine in the renal total lipids. The significant decrease of phosphatidyl ethanolamine in the kidney phospholipids appears to implicate this phospholipid to a greater extent than phosphatidyl choline in the etiology of the hemorrhagic syndrome.     

Quantitative analysis of the phospholipids of some marine bioluminescent bacteria

Quantitative analyses of the phospholipids of three strains of marine bioluminescent bacteria were carried out after separation by two-dimensional thin layer chromatography. The phospholipids of all three species consisted of about 75% phosphatidyl ethanolamine, 13% phosphatidyl glycerol and 7% cardiolipin. The composition was only slightly affected by drastic changes in the growth conditions. One of the species contained poly-β-hydroxybutyrate. The fatty acids of another species contained principally straight and branched-chain 16 and 18 carbon fatty acids. No clue as to the nature of the elusive “aldehyde factor” of bacterial bioluminescence was found by analysis of aldehyde deficient mutants, indicating that possibly the factor is not a major phospholipid of these bacteria.     

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.     

Studies on the glycolipids and phospholipids of immature soybeans

The lipid of immature soybeans was extracted with chloroform-methanol and fractions containing the glycolipids and phospholipids were separated by column chromatography. Phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidic acid (PA) phosphatidyl glycerol (PG), n-acyl phosphatidyl ethanolamine (APE) and sulfolipid (SL) were identified by thin layer chromatography (TLC). Sterol glucoside (SG), esterified sterol glucoside (ESG), digalactosyl diglyceride (DGDG) and cerebrosides (CE) were isolated by TLC and identified by color reactions, chemical degradation and spectral analysis.     

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.     

Species variations in phospholipid class distribution of organs: II. Heart and skeletal muscle

Total lipid, lipid phosphorus and phospholipid class distribution were determined for heart and skeletal muscles from five vertebrates (human, bovine, rat, mouse and frog) and skeletal muscles from four invertebrates (lobster, abalone, scallop and sea urchin). The precision of the analytical method [separation by two-dimensional thin layer chromatography (TLC) and phosphorus analysis of spots] was demonstrated by small values for standard deviations. Accuracy of spot identification and analytical values was insured by comparison with results obtained by TLC of triethylaminoethyl cellulose column chromatographic fractions. Values for total lipid, total phospholipid and phospholipid class distribution of heart and skeletal muscles from the five vertebrate species showed essentially the same variability observed for the same organ from different animals of one species (rat). The data indicate that, among vertebrates there is little or no variability for phospholipid class distribution in muscle membranes in agreement with data for other organs and subcellular particulates presented in other reports. Invertebrate skeletal muscles were found to differ qualitatively and/or quantitatively from those of vertebrates. In one species (sea urchin), ceramide phosphorylethanolamine was the only sphingolipid, sphingomyelin characteristic of vertebrates being absent. In two species (abalone and scallop) ceramide aminoethylphosphonate was present and sphingomyelin was absent. In one (lobster), sphingomyelin was the only sphingolipid. Quantitatively, higher levels of sphingolipid and phosphatidyl serine and lower levels of phosphatidyl inositol were found in invertebrate skeletal muscle. The significance of the data is discussed in relation to subcellular particulate lipid class composition.     

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 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.     

A comparative study of the lipids of chylomicron membrane and fat core and of the lymph serum of dogs

Thoracic lymph was collected from 13 dogs fed corn oil and butterfat. The chylomicrons were isolated by centrifugation. The lipid composition of the fat core and the membrane of the chylomicron was compared to that of the surrounding lymph serum. The fat cores contained 90–96% triglyceride, 0.7–1.9% free cholesterol, 0.2–0.5% steryl ester, 0.9–3.5% free fatty acid and 1.4–6.1% diglyceride, but no phospholipid. The lipids of the membranes contained 58–75% phospholipid, 20–35% triglyceride, 2–5% free cholesterol, 1–2% free fatty acid, and 2–3% diglyceride, but little or no steryl ester. The membrane phospholipids were made up of 70–90% lecithin, 5–20% phosphatidyl ethanolamine, and 1–3% each of lysolecithin and sphingomyelin. The lymph serum contained 24–47% of total lipid as phospholipid, of which 70–92% was lecithin; the phosphatidyl ethanolamine, lysolecithin and sphingomyelin also present contributed 1–10% each. The neutral lipids of the lymph serum contained 49–75% triglyceride, 2–15% free cholesterol, 6–23% esterified cholesterol, 10–33% free fatty acid and 1–6% diglyceride. Alterations in dietary fat, or plant sterol supplementation led to lesser changes in the lipids of the chylomicron membranes than in the lipids of any other lymph fraction. The least variation was seen in the phospholipids. Taken in part from a PhD Thesis submitted by T. C. Huang to Queen's University, Kingston, Canada, in April 1965. Presented at the AOCS 56th Spring Meeting, Houston, May 1965.