Quantitative determination of alk-1-enyl- and alkyl-glyceryl ethers in neutral lipids and phospholipids

A quantitative method for the simultaneous determination of alk-l-enyl- and alkyl-glyceryl ethers is described. Complete hydrogenolysis of carboxylate and phosphate esters of neutral lipids and phospholipids was achieved with lithium aluminum hydride. The hydrogenolysis products of the glyceryl ether containing lipids, alk-l-enyl- and alkyl-glyceryl ethers and alcohols, were identified by thin-layer chromatography (TLC), gas-liquid chromatography (GLC), and infrared spectroscopy. The alk-l-enyl- and alkyl-glyceryl ethers were quantitated by TLC photodensitometry. The specificity of this method can also be extended when used in conjunction with GLC, ion-complexing TLC, zonal scanning, and autoradiography to study composition, isomeric form, and the biosynthesis of glyceryl ethers in neutral and phospholipids. The percentage of alk-l-enyl- and alkyl-glyceryl ethers in bothtthe neutral lipids and phospholipids of various rat tissues was determined by the described method. Glyceryl ether glycerides represent 0.3–1.2% of the total neutral lipids whereas the glyceryl ether phosphatides of brain, heart, marrow, muscle, and spleen represent 4.5–12.0% of their total phospholipids. Higher concentrations of glyceryl alkyl than of alk-l-enyl ethers were found in the neutral lipids whereas the glyceryl alk-l-enyl ethers were found to predominate in the phospholipids. Presented at the AOCS Meeting, Chicago, October 1967. Under contract with the U. S. Atomic Energy Commission.     

Ether glycerophospholipids of gills of two pacific crabs,Cancer antennarius andPortunus xantusi

Phospholipids and sterols constituted 70 and 20%, respectively, of the total lipids of the gills of two crabs,Cancer antennarius andPortunus xantusi. Phosphatidylcholine (46–55% of the total phospholipid phosphorous) and phosphatidylethanolamine (24–25%) were the principal phospholipids present. In both species 1′-alkenyl glycerols were present in about 20% of the phospholipid molecules but were not detected in the neutral gill lipids. The total ether phospholipids ofC. antennarius gills contained 62% 1-(1′-alkenyl) groups, with the remainder probably being 1-alkyl moieties. Total gill plasmalogen contents were in the range of 163–184 μmol/g lipid, 82–87% of which was in the phosphatidylethanolamine fraction in both crab species.     

The lipid components of white potato tubers (Solanum tuberosum)

Four Canadian varieties of potatoes were examined for their lipid composition. Lipids, extracted with chloroformmethanol, were shown by TLC and column chromatography to consist of 16.5% neutral lipids, 45.5% phospholipids and 38.1% glycolipids. Among the phospholipids and glycolipids, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, the galactolipids and the sterol glucosides were the major lipids. The predominant fatty acids were palmitic (19.5%), linoleic (44.8%) and linolenic (30.4%, in Kennebec). Analyses of the fatty acids of stored potatoes showed a marked decrease in linoleic acid and an increase in linolenic acid, in the Irish Cobbler and Sebago potatoes. β-sitosterol comprised 85.0% of total sterols. Nearly half of the carotenoids was lutein (xanthophyll), the others being α-carotene, β-carotene, an unidentified pigment and lutein epoxide. Contribution No. 101 of the Food Research Institute, Canada Department of Agriculture.     

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.     

Evidence for diplasmalogen as the major component of rabbit sperm phosphatidylethanolamine

The question of whether diplasmalogens [1,2-di(O-1′-alkenyl) phosphatidyl derivatives] make up part of the plasmalogen component of cell phospholipids was examined using rabbit epididymal spermatozoa. These cells are readily obtained as a highly homogeneous suspension and long have been known to have high plasmalogen content. Phospholipids were determined by thin layer chromatography (TLC) with CuSO₄ staining. Plasmalogens were determined by hydrolysis of the phospholipids with TCA/HCl, followed by TLC and CuSO₄ staining. Ethanolamine derivatives were determined by ninhydrin. The phosphatidylethanolamine (PE) content of these cells was 29±2 μg/10⁸ cells, 90% of which was assayed as diplasmalogen and 10% as diacyl PE. No monoplasmalogen could be detected. The presence of diplasmalogen as the major component of PE was given further support from infrared and proton nuclear magnetic resonance (¹H-NMR) spectroscopy, which showed the presence of O-1′-alkenyl substituents but near absence of O-acyl substituents. The phosphatidylcholine (PC) content of the cells was 104±5 μ/10⁸ cells, of which 50% was monoplasmalogen with the 1′-alkenyl group on the 2 position of the glycerol moiety. No diplasmalogen was found in PC. The other phospholipids in rabbit sperm were phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SP) and lysophosphatidylcholine (LPC). Phosphatidylserine (PS) and phosphatidylinositol (PI) were present at the limits of detectability of the TLC method. None of these phospholipids contained plasmalogen. The PE component of rabbit sperm phospholipids appears to differ from that of the other cells in having the previously unreported diplasmalogen as its major constituent.     

Quantitative analysis of lipid classes by liquid chromatography via a flame ionization detector

A method is described for the direct quantitative analysis of the lipid classes of mammalian tissue lipids using high performance liquid chromatography (HPLC) with a flame ionization detector (FID). The lipid is extracted from the tissue with chloroform/methanol after deactivation of hydrolytic enzymes and removal of nonlipid substances by extraction with hot dilute acetic acid (0.05N). Separation of the lipid classes is performed with a column (45 cm × 0.2 cm id) of 8 μm silica (Spherisorb, Phase Sep, Hauppague, NY) treated with concentrated ammonium hydroxide at a solvent flow rate of 0.5 ml/min, which requires a pressure of ca. 900 psi. Cholesteryl esters (CE) and triglycerides (TG) are eluted first with Skellysolve B/methylene chloride (1∶1, v/v); cholesterol (CH) is eluted with chloroform/methylene chloride (1∶2, v/v) and the phospholipids with methanol containing 6% ammonium hydroxide added to the latter solvent in a linear gradient. The neutral lipids are eluted in ca. 12 min and the phospholipids in an additional 30 min. The relative amount of each lipid class was determined from standard curves of the peak areas obtained according to response factors using erucyl alcohol as an internal standard. The method was applied to samples of kidney, liver and serum of rats. Duplicate analyses were generally within ca. 1.0% and good agreement was obtained in the analysis of the lipid classes of Azolectin and liver mitochondria lipid compared to thin layer chromatography (TLC) via photodensitometry of charred spots or phosphorus analysis of recovered phospholipids.     

Synthesis of spin-labeled neutral lipids: Nitroxyl derivatives of triglycerides and sterol esters

Methods for the preparation of useful spin-labeled neutral lipids are described. A spin-labeled triglyceride has been prepared by acylation of 1,3-distearoylglycerol with stearic acid anhydride bearing the 4′,4′-dimethyloxazolidine-N-oxyl ring at carbon-12. The same fatty acid anhydride has been used to acylate the 3-hydroxy group of cholesterol to obtain a cholesteryl ester with the nitroxyl function in the fatty acyl chain. The 4′,4′-dimethyloxazolidinyl-1-oxyl derivative of 5α-an drostan-3-one-17β-ol has been esterified with stearic acid anhydride to obtain a steroid ester with the paramagnetic center in the steroid nucleus.     

Lipid metabolic interrelationships and phospholipase activity in gustatory epithelium ofictalurus punctatus in vitro

The catfish,Ictalurus punctatus, is an important model for studying the biochemical mechanisms of taste at the peripheral level. The type, amount and metabolic activity of the lipids within this tissue play important roles in taste transduction by forming the matrix in which the receptors for taste stimuli are imbedded and by acting as precursors to second messengers. The metabolic interconversions that occur among the lipids on the taste organ (barbels) of this animal are reported here. When sodium [³²P]phosphate was incubated with minced pieces of epithelium from the taste organ ofI. punctatus, phospholipids became labeled. Maximal incorporation occurred near 20 min for lysophosphatidylcholines (LPC),phosphatidylcholines (PC) and phosphatidylinositols (PI). The phosphatidylethanolamines (PE) and phosphatidylserines (PS) became labeled more slowly. The label in LPC and PC declined from 20 min to 120 min, while that of the other fractions increased or was stable over the 20–120 min time period. Upon addition of 1,2-di-[1′-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine to the medium,¹⁴C was found within minutes in all of the phospholipids assayed. The amount of label incorporated increased with time, with maximum labeling for all phospholipids occurring at 15 min. However,¹⁴C appeared predominantly first (by 5 min) in a neutral lipid fraction (fraction AG, consisting of free fatty acids, mono- and diglycerides, triglycerides and methyl esters), then declined rapidly as the phospholipids gradually incorporated more label. Within minutes of addition of 1-[1′-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine (lysophosphatidylcholine) the¹⁴C-label was detected in the neutral lipid fraction AG, then in the PC fraction, and later in the other phospholipids. The PC fraction was maximally labeled by 40 min. Using the appropriate radiolabeled substrates, lysophosphatidylcholine phospholipase A₁ and phosphatidylcholine phospholipase D activities were detected in this tissue. Very low activity of a phosphatidylcholine phospholipase A₂ was observed. The experiments indicate that there are active and rapid exchange, degradation, synthesis and scavenger pathways of phospholipids in the taste organ of this animal, and suggest that phospholipases A₁ and D-type activities are primarily responsible for the rapid breakdown of LPC and PC.     

Methoden zur Phosphatidanalytik am Beispiel der Lipide aus Seren und Eigelb

Techniques of Phospholipid Analyses Applied to Sera and Egg Yolk Thin layer (TLC) and gas-liquid-chromatography (GLC) were used to analyse phospholipids and diglycerides prepared from phospholipids. TLC of lipids is carried out on silica gel and for diglycerides on silica gel impregnated with 10% AgNO₃. Fatty acids of individual lipids were identified by GLC. The recovery of lipids was evaluated after chromatography of phospholipids and diglycerides and after digestion with phospholipase-C from Bacillus cereus. Two techniques of acetylation with two different catalysts are compared. The calculations were referred to phosphorus or glycerol in lipids. The precise amount of phospholipids was determined after charring as inorganic phosphorus. Diglycerides were assayed after saponification by an enzymatic determination of glycerol. The reliability of some lipid techniques could be shown. The values of phospholipids for egg yolk and sera are in correspondence to literature.     

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.     

Separation and quantitation of phospholipids in animal tissues by latroscan TLC/FID

A procedure has been developed to separate and quantitate phospholipids, including phosphatidylinositol and phosphatidylserine, from animal tissues by means of the Iatroscan TLC/FID technique. The method is based on the use of 0.01 M oxalic acid impregnated Chromarods-SII and stepwise resolution of the phospholipids in the presence of 1,2-dipalmitoyl-sn-glycero-3-phospho (N,N-dimethylethanolamine) as internal standard. To remove the neutral lipids, the rods are initially developed in a nonpolar solvent mixture followed by partial scanning. Next, the rods are impregnated with oxalic acid, developed twice in CHCl₃/CH₃OH/CH₃COOH/HCOOH/H₂O (80∶35∶2∶1∶3, v/v/v/v/v) and partially scanned for measuring lysophosphatidylcholine, sphingomyelin and phosphatidylcholine. The subsequent step involves double development in CHCl₃/CH₃OH/30% NH₄OH (60∶35∶0.9, v/v/v) to resolve cardiolipin, internal standard, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid. For each phospholipid a linear calibration curve with a highly significant correlation coefficient was obtained. However, the calibration lines extrapolated to negative intercepts on the ordinate, indicating declining sensitivity at low phospholipid loads.     

Lipid composition of rice (Oryza sativa L.) bran

The composition of lipids of bran from three varieties of rice is reported. Lipids extracted amounted to 21.9–23.0% of the bran dry weight and consisted of 88.1–89.2% neutral lipids, 6.3–7.0% glycolipids and 4.5–4.9% phospholipids. Neutral lipids consisted mostly of triacylglycerols (83.0–85.5%), monoacylglycerols (5.9–6.8%) and small amounts of diacylglycerols, sterols and free fatty acids. Three glycolipids and eight phospholipids were separated and characterized. Acylated steryl glucoside and digalactosyldiacylglycerol were the main glycolipids, while monogalactosylmonoacylglycerol was present in small amounts. The major phospholipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidic acid. Phosphatidylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine and acyl-phosphatidylethanolamine were present in small quantities.     

Polyunsaturated fatty acids and neutral lipids in developing larvae of the oyster,Crassostrea virginica

The fatty acid composition of oyster larvae at various stages, as well as of the algal diet, were determined by gas liquid chromatography (GC). Saturated fatty acids are the major fatty acid components in all larval stages and account for 34–62%, 30–35% and 35–81% of the neutral, polar and total lipids of algal-fed larvae respectively. Weight percentage of saturated fatty acid in “starved” larvae was consistently higher (63–81%) during the whole period. The total polyunsaturated fatty acids were higher in the polar lipids than in the neutral lipids. The concentration of the ω3 fatty acids also was comparatively higher in the polar lipids than in the neutral lipids. In the total and neutral lipid fractions, the weight percentage of polyunsaturated and ω3 fatty acids was higher in the eyed than in the pre-eyed (pediveliger) larvae. Eicosapentaenoic acid (20∶5ω3) and 22∶6ω3 were not detected in lipids of “starved” and young larvae. There was an accumulation of 20∶5ω3, 22∶6ω3, and total ω3 fatty acids in the older larvae. Lipid classes were separated by thin layer chromatography (TLC). There was no qualitative change in lipid composition during larval development, but a marked increased of triacylglycerol in larvae up to the stage of maturation in algae-fed larvae. Contribution number 1195 of the Virginia Institute of Marine Science, Gloucester Point, VA 23062     

Analyses of lipids and fatty acids in ripe roes of some Northwest European marine fish

Lipid class analyses and fatty acid analyses of neutral and polar lipids were carried out on ripe roes of herring, cod, haddock, whiting, saithe, sand eel and capelin. Total lipid was 10–26% of roe dry weight. The species with the highest total lipid, sand eel and capelin, also had the highest percentage of neutral lipid in total lipid, 77% and 49% respectively. In the other species, phospholipids accounted for 62–77% of roe total lipid. Both the neutral lipids, and especially the phospholipids, of all species were very unsaturated because of high concentrations of (n−3) polyunsaturated fatty acids (PUFA), frequently amounting to 50% of the total egg lipid. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) had similar fatty acid compositions in all species, with an average ratio (n−3)/(n−6) of ca. 20∶1. Phosphatidylinositol (PI) consistently had high concentrations of 18∶0 and 20∶4 (n−6) with an average ratio of (n−3)/(n−6) of 1.8∶1. Requirements for high levels of (n−3) PUFA in the embryonic and early larval development stages of marine fish are suggested as is a special role for the 20∶4(n−6) in PI.     

Quantitative analysis of lipids by thin-layer chromatography

A procedure is described for the quantitative analysis of neutral and phospholipids by thinlayer chromatography (TLC) employing densitometry. The chromatophates are prepared with the usual solvent systems. The spots are charred under standard conditions and analyzed with a Photovolt Corp. densitometer equipped with a special stage designed for holding 20×20 cm chromatoplates. Each spot on the chromatoplate gives a peak of density values which is used for quantitative analysis. Radioactive lipids are analyzed by autoradiography by the densitometry of radiograms of chromatoplates developed from X-ray films. The precision of the method is demonstrated on model mixtures of mono, di-and triglycerides, neural and phospholipids and C¹⁴ labeled lipids. Results of the analysis of several samples of rat liver lipids compared closely to those obtained by silicic acid column chromatography. Supported in part by U. S. Public Health Service. Research Grant HE-05375.     

Varietal difference in lipid content and fatty acid composition of highbush blueberries

Lipids from five cultivars of highbush blueberries (Vaccinium corymbosum L.) were extracted and fractionated into neutral lipids (60–66%), glycolipids (20–22%) and phospholipids (14–18%). The major fatty acids in all fractions were palmitic (16∶0), oleic (18∶1), linoleic (18∶2), and linolenic (18∶3) acids. All lipid classes had a large concentration of C₁₈ polyunsaturated acids (84–92%), indicating that blueberries are a rich source of linoleic and linolenic acids. Changes in the fatty acid composition of neutral lipids and phospholipids were not significantly different among the five cultivars, but significant differences were noted in the ratios of linoleic and linolenic acids in the glycolipids fraction.     

Quantitating heart lipids: Comparison of results obtained using the Iatroscan method with those from phosphorus and gas chromatographic techniques

The precision and accuracy of the Iatroscan method was evaluated by comparing the results obtained with established phosphorus and gas chromatographic techniques. A complete lipid class analysis of rat heart lipids was chosen in order to evaluate the performance of the Iatroscan method for biological samples which contained both neutral lipids and phospholipids. A partial scan and repeat development with chloroform/methanol/water (68.5∶29∶2.5) was introduced to achieve consistently good separations of the phospholipids on the Chromarods in the Iatroscan method. The results showed that the precision of the Iatroscan method for some lipid classes was comparable to that of phosphorus or gas chromatographic techniques, while for other lipid classes it was lower. Compared to the data obtained using the phosphorus method, the Iatroscan data were generally similar, while the gas chromatographic method generally gave lower values. These findings, together with the advantages of time required for analysis, size of sample, and universality of detection, suggest that the Iatroscan is a valuable complementary method for complex lipid analyses.     

In vitro incorporation of 1-14C nonanal-9-oic acid into plasma and human red blood cells lipids

Nonanal-9-oic acid is incorporated principally into plasma phospholipids, whereas oleic acid is incorporated into red cells. This incorporation does not require the presence of adenosine 5′-triphosphate and Coenzyme A and is carried out in the absence of red cells. the incorporation of nonanal-9-oic acid in blood lipids takes place in the first 10 min of incubation.     

Lipid composition of normal male rat islets

Lipid composition was studied in fresh isolated isolets from normal male rats. Extractable lipids represent 1856 μg per mg islet protein. In such extracts, phospholipids and neutral lipids reprsent 13.5% and 86.5%, respectively. Phosphatidylcholine (45.8%) and phosphatidyl-ethanolamine (20.6%) were the major components of the phospholipid fraction, and phosphatidylinositol (8.9%) was the minor component. Esterified cholesterol (38.5%), cholesterol (25.5%) and free fatty acids (24.4%) were the major components of the neutral lipid fraction. Fatty major components of the neutral lipid fraction. Fatty acids esterified to phospholipids account for 619.7 pmol/islet, and, 2710 pmol/islet were esterified to neutral lipids. In the phospholipid fraction, saturated and unsaturated fatty acids were in a similar proportion. Conversely, in the neutral lipids, two-thirds of the fatty acids were unsaturated. The ω6 family was the main component of the phospholipid unsaturated fatty acids. In the ω6 and ω3 families, the long-chain fatty acids represent the main components. In the neutral lipid fraction, a different percentage of each family was found: ω3>ω6>ω9. The long-chain polyunsaturated fatty acids were also predominant species in the ω6 and ω3 families. Further studies on the lipid composition of islets, obtained from rats with normal and altered islet functions, could provide new insights into the knowledge of the mechanism of insulin secretion.     

Incorporation of lipids labeled with various fatty acids into the cytoskeleton of aggregating platelets

Earlier studies showed that during the first 20 to 25 seconds of aggregation induced by thrombin (0.1 U/mL) or adenosine diphosphate (ADP) (2μM) of rabbit or human platelets prelabeled with [³H]palmitic acid, labeled lipid became associated with the cytoskeleton (isolated after lysis with 1% Triton X-100, 5 mM EGTA [ethylene glycol-bis-(β-aminoethyl ether(N,N,N′,N′-tetraacetic acid] in the presence of 0.5 mM leupeptin and 50 mM benzamidine). In comparison with labeled lipid in intact platelets, the labeled lipid that was associated with the cytoskeleton was enriched in phospholipids and ceramide. To determine whether these effects were specific for lipids labeled with palmitic acid, we studied rabbit platelets in which lipids had been labeled by incubation of the platelets with pairs of¹⁴C- or³H-labeled palmitic, stearic, arachidonic, and linoleic acids. Examination of the distribution of label among the lipid classes of intact platelets showed that phospholipids contained most of the label. Under the conditions of limited, thrombin-induced aggregation used, labeled lipids were not lost from the platelets and the distribution of label among the lipid classes was essentially unchanged. There were major differences in the incorporation of labeled lipids into the cytoskeleton. The greatest incorporation (2.1 to 2.8% of the label in the platelets) was observed with palmitic acid-labeled lipids; by direct comparison, only 44% as much of the label of stearic acid-labeled lipids, 21% as much of the label of linoleic acid-labeled lipids, and only 6% as much of the label of arachidonic acid-labeled lipids was incorporated into the cytoskeleton. Thus the pool of phospholipid that is readily labeled with arachidonic acid appears to be selectively excluded from the cytoskeleton. Also noteworthy is the 4- to 5-fold enrichment of the cytoskeleton with labeled ceramide; an average of 16% of the label from stearic acid in the cytoskeleton was in ceramide. We suggest that ceramide and phospholipids that are readily labeled with saturated fatty acids are selectively incorporated into the cytoskeleton during the early stages of aggregation and may be specifically associated with the points of contact between platelets.