Lipid metabolism in brain tissue explants

Abstract— Tissue explants from frontal lobes of rat brain were used for the study of cerebral fatty acid metabolism. After tissues had been maintained in serum-supplemented medium, a lipid-free medium was substituted and metabolic studies were carried out. Under these conditions explants continued to take up lipid precursors for at least 48 h, as judged by incorporation of dl -[2-¹⁴C]mevalonic acid into cellular lipids. [l-¹⁴C]Stearic acid and [l-¹⁴C]palmitic acid were bound to cells as the free fatty acids, or incorporated into neutral lipids (particularly triglycerides), glycolipids and phospholipids. In the galactolipid fraction, cerebrosides were the principal radioactive lipids. Choline phosphoglycerides, ethanolamine phosphoglycerides, inositol phosphoglycerides and serine phosphoglycerides were the principal radioactive phospholipids. Fatty acids were incorporated into cellular lipids either unchanged or after desaturation, chain elongation, or both. Maximum incorporation of stearate occurred in tissues derived from 3-day-old animals. With increasing age the uptake of fatty acid dropped sharply. When the labelling of lipids as a function of time was followed in 3-day-old animals, triglycerides and choline phosphoglycerides were the first fractions to take up labelled stearate. Labelling of cerebrosides occurred slowly, only becoming evident after 24 h. These studies exemplify the usefulness of tissue explants for prolonged metabolic studies in normal and pathological specimens of brain.     

DIFFERENTIAL DISTRIBUTION OF [U-14C]GLUCOSE AND [U-14C]GLYCEROL AMONG MOLECULAR SPECIES OF PHOSPHATIDYL CHOLINE,PHOSPHATIDYL ETHANOLAMINE AND 1,2-DIACYLGLYCEROL IN RABBIT BRAIN12

Specific radioactivities of molecular species of phosphatidyl choline(PC), phosphatidyl ethanolamine(PE) and 1,2-diacylglycerol were determined in rabbit brain 15 and 30 min after intraventricular injection of 10OpCi of either [U-¹⁴C]glucose or [U-¹⁴C]glycerol. The rate of de nouo synthesis of glycerophospholipids and their molecular species could be determined after glycerol labelling, since 94.0–99.7% of ¹⁴C activity was recovered in glyceryl moieties of brain lipids. After injection of glucose radioactivity was measured in both glyccrol and acyl residues of lipids. High incorporation rates were measured in species of PC, PE and 1,2-diacylglycerol with oleic acid in position 2 and with palmitic, stearic or oleic acids in position 1. The conclusion may therefore be drawn that these molecular species were preferably synthesized de novo by selective acylation of glycerol 3-phosphate. The lowest specific activities were observed for 1,2-dipalmitoyl- and l-stearoyl-2- arachidonoyl-glycerol, -PC and -PE. These turnover rates point to incorporation of arachidonate, and probably also of palmitate in dipalmitoyl-PC, amounting to 20% of total PC, via deacylation-acylation- cycle.     

Direct effects of temperature on phospholipid and polyunsaturated fatty acid metabolism in isolated brain cells from rainbow trout, Oncorhynchus mykiss.

1. The direct effects of temperature on the metabolism of [1-14C]18:2(n-6), [1-14C]18:3(n-3), [1-14C]20:4(n-6) and [1-14C]20:5(n-3) were studied in isolated brain cells from rainbow trout, Oncorhynchus mykiss. 2. Recovery of radioactivity from all the polyunsaturated fatty acids (PUFA) in total lipid was significantly greater at 5 and 15 degrees C than at 25 degrees C. 3. The lower incubation temperatures decreased the relative net incorporation of all the 14C-labelled PUFA into phosphatidylcholine (PC) and increased the relative incorporation of the PUFA into the other phosphoglycerides, especially phosphatidylethanolamine (PE). 4. The effects on PC were generally more significant between 25 and 15 degrees C, whereas the effects on PE were generally significant both between 25 and 15 degrees C and between 15 and 5 degrees C. 5. This suggests that the lysophospholipid acyltransferases responsible for the incorporation of PUFA into different phosphoglycerides may have differential sensitivities to temperature. 6. In contrast, the acyltransferase activities showed fatty acyl preferences that were independent of temperature. 7. Although a trend towards decreased activity at 5 degrees C was apparent, temperature generally had little significant effect on the relative percentages of the PUFA metabolized via the desaturase pathways.     

Metabolism of the ethanolamine phosphoglycerides of mouse brain myelin and microsomes

Abstract— Three groups of six mice each were killed 1, 4 and 7 days after an intracerebral injection of [1,2-¹⁴C]ethanolamine. The specific radioactivities of the acid-labile ethanolamine phosphoglycerides (ethanolamine plasmalogens) and of the acid-stable ethanolamine phosphoglycerides (diacyl and alkyl acyl glycerophosphoryletholamines) from myelin and microsomal fractions were determined. All of these brain ethanolamine phosphoglycerides turn over rapidly with an apparent half-life of less than 3 days. The biosynthesis of alkenyl acyl glycerophosphorylethanolamines from diacyl glycerophosphorylethanolamines in mouse brain myelin or microsomes is unlikely.     

Further studies on the metabolism of phospholipids inStreptomyces griseus: Turnover of phospholipid moieties

Phospholipid turnover inStreptomyces griseus was studied by pulse-chase techniques using 1-[¹⁴C]sodium acetate and [U-¹⁴C]glucose. Different phospholipids and their individual moieties were found to have different turnover rates. The moieties of inositol-containing phospholipids exhibited the fastest turnover rates among the major phospholipids, while only fatty acyl moieties of phosphatidylethanolamine showed rapid turnover. Cardiolipin did not show any significant turnover with both precursors.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Turnover of Axonally Transported Phospholipids in Nerve Endings of Retinal Ganglion Cells

We have investigated the metabolic turnover of axonally transported phospholipids in myelinated axons (optic tract) and nerve endings (superior colliculus) of retinal ganglion cells. One week following intraocular injection of [2-3H]glycerol, turnover rates for individual phospholipid classes in the retina (which contains a number of other cell types in addition to the ganglion cells) were all very similar to each other, with apparent half-lives of approximately 7 days. Apparent half-lives of labeled phospholipids in superior colliculus (presumably primarily in retinal ganglion cell nerve endings) were 10 days for both choline and inositol phosphoglycerides and 13 days for both serine and diacylethanolamine phosphoglycerides. Subcellular fractionation data obtained from superior colliculus at various times after injection suggested that apparent turnover rates determined for nerve ending phospholipids probably were not significantly affected by transfer of axonally transported 3H lipids into myelin. Apparent half-lives for phospholipids in optic tract were somewhat longer than in superior colliculus, ranging from 11 to 18 days. The slower turnover rates in optic tract may, in part, reflect the transfer of some axonal lipids to the more metabolically stable pool of lipids in the myelin ensheathing the retinal ganglion cell axons. In both optic tract and superior colliculus, apparent half-lives for axonally transported phospholipids labeled with [32P]phosphate were only slightly longer than for [2-3H]glycerol, while those for [14C]choline and [3H]acetate were markedly longer, indicating differing degrees of metabolic conservation or reutilization of these precursors relative to glycerol.     

Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston

Erucic acid (22:1) was chosen as a marker to study triacylglycerol (TAG) biosynthesis in a Brassica napus L. cv Reston microspore-derived (MD) embryo culture system. TAGs accumulating during embryo development exhibited changes in acyl composition similar to those observed in developing zygotic embryos of the same cv, particularly with respect to erucic and eicosenoic acids. However, MD embryos showed a much higher rate of incorporation of ¹⁴C-erucoyl moieties into TAGs in vitro than zygotic embryos. Homogenates of early-late cotyledonary stage MD embryos (14-29 days in culture) were assessed for the ability to incorporate 22:1 and 18:1 (oleoyl) moieties into glycerolipids. In the presence of [1-¹⁴C]22:1-coenzyme A (CoA) and various acyl acceptors, including glycerol-3-phosphate (G-3-P), radiolabeled erucoyl moieties were rapidly incorporated into the TAG fraction, but virtually excluded from other Kennedy Pathway intermediates as well as complex polar lipids. This pattern of erucoyl incorporation was unchanged during time course experiments or upon incubation of homogenates with chemicals known to inhibit Kennedy Pathway enzymes. In marked contrast, parallel experiments conducted using [1-¹⁴C]18:1-CoA and G-3-P indicated that ¹⁴C oleoyl moieties were incorporated into lyso-phosphatidic acids, phosphatidic acids, diacylglycerols, and TAGs of the Kennedy Pathway, as well as other complex polar lipids, such as phosphatidylcholines and phosphatidylethanolamines. When supplied with l-[2-³H(N)]G-3-P and [1-¹⁴C]22:1-CoA, the radiolabeled TAG pool contained both isotopes, indicating G-3-P to be a true acceptor of erucoyl moieties. Radio-high-performance liquid chromatography, argentation thin-layer chromatography/gas chromatography-mass spectrometry, and stereospecific analyses of radiolabeled TAGs indicated that 22:1 was selectively incorporated into the sn-3 position by a highly active diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), while oleoyl moieties were inserted into the sn-1 and sn-2 positions. In the presence of sn-1,2-dierucin and [1-¹⁴C]22:1-CoA, homogenates and microsomal preparations were able to produce radiolabeled trierucin, a TAG not found endogenously in this species. A 105,000g pellet fraction contained 22:1-CoA:DGAT exhibiting the highest specific activity. The rate of 22:1-CoA:DGAT activity in vitro could more than account for the maximal rate of TAG biosynthesis observed in vivo during embryo development. In double label experiments, G-3-P was shown to stimulate the conversion of [³H]phosphatidylcholines to [³H]diacylglycerols, which subsequently acted as acceptors for ¹⁴C erucoyl moieties. In vitro, 22:1 moieties did not enter the sn-1 position of TAGs by a postsynthetic modification or transacylation of preformed TAGs.     

A COMPARATIVE STUDY OF THE METABOLISM OF DE NOVO SYNTHESIZED FATTY ACIDS FROM ACETATE AND GLUCOSE, AND EXOGENOUS FATTY ACIDS, IN SLICES OF RABBIT CEREBRAL CORTEX DURING DEVELOPMENT

Slices of rabbit cerebral cortex, from the foetal stage to the adult have been used to compare lipid synthesis from fatty acids synthesized de novo from [U-¹⁴C]glucose and [1-¹⁴C]acetate, with lipid synthesis from exogenous albumin-bound [1-¹⁴C]palmitate. Incorporation into cellular lipid has been determined in terms of DNA, protein, wet wt. of tissue and wet weight of whole brain. On a wet wt. basis, maximum incorporation of glucose carbon into lipid occurred in the foetal brain while lipid synthesis from acetate and palmitate was maximum at 4–14 days after birth. Glucose and acetate were incorporated into a diversity of lipids (with increasing amounts of phosphatidylcholine synthesized during maturation), while palmitate was incorporated into the free fatty acid and triglyceride fractions. A greater proportion of acetate was incorporated into fatty acids of chain-length longer than C16 compared with the incorporation of palmitate. However, on a molar basis de novo synthesized and exogenous palmitate were elongated, desaturated and incorporated into phospholipids at a similar rate, while exogenous palmitate was incorporated to a greater extent than de nova synthesized fatty acid into the triglyceride fraction. This difference in metabolism may be due to the different size of the non-esterified fatty acid pool in the two situations. At the period of their most active formation, the very long-chain fatty acids may be synthesized from a pool of the C18 series of fatty acids (saturated and monoenoic) not in equilibrium with the bulk of C₁₈ acids in cerebral lipids. This could be a pool of acyl groups derived from ethanolamine phospholipids.     

TURNOVER OF PHOSPHATIDYLCHOLINE IN MICROSOMES AND MYELIN IN BRAINS OF YOUNG AND ADULT RATS

We have tested the hypothesis that the turnover of phosphatidylcholine in subcellular fractions of rat brain is a function of the age at which this lipid is deposited. Rats, 60 days of age, were injected intracranially with [2-³H]glycerol and either [methyl-¹⁴C]choline (to label the base moiety) or [U-¹⁴C]glucose (to label acyl moieties). Littermates were killed up to 90 days after injection and brain microsomes and myelin isolated. Lipids were extracted and the phosphatidylcholine was isolated by 2-dimensional TLC and hydrolyzed to its constituent moieties. The ³H in the glycerol backbone and ¹⁴C in the choline or acyl residues was quantitated. The microsomal and myelin ³H/¹⁴C ratios decreased with time with either set of precursors, indicating that labeled choline and acyl moieties were reutilized more efficiently than the glycerol backbone. The various precursors exhibited first order decay curves with half-lives for the glycerol backbone of 6 and 11 days for the microsomal and myelin fractions respectively. These results contrast with those previously obtained with identical experimental procedures when 17-day-old animals were injected. In that study, although much of the phosphatidylcholine turned over rapidly as for the older animals, by 2 weeks after injection most of the remaining phosphatidylcholine was turning over more slowly with a half-life of 13 and 25 days for microsomes and myelin respectively (Miller et al., 1977). The base and acyl moieties also had a corresponding shorter half-life in older animals relative to the slow turnover phase in younger rats.     

Dependence with nutritional status of the ethanol effects on fatty acid metabolism in rat hepatocytes

1. The effects of ethanol on fatty acid synthesis, esterification and oxidation were studied in hepatocytes isolated from fed and 24 hr fasted rats. 2. [3H]H2O was preferentially incorporated into the glycerol backbone of triglycerides and phospholipids. Addition of ethanol markedly increased the incorporation of this label in both classes of glycerolipids; the increase was higher in fasted rat hepatocytes, both in the glycerol backbone and acyl groups of glycerolipids. 3. Ethanol increased [U-14C]palmitate incorporation into triglycerides only in hepatocytes from fasted rats. 4. [14C]CO2 and total acid soluble product formation from [1-14C]palmitate resulted inhibited by ethanol both in the fed and the fasted state.     

Modification of glycerolipid metabolism in L-M fibroblasts by an unnatural amino-alcohol, N-isopropylethanolamine.

The unnatural amino-alcohol, N-isopropylethanolamine, is incorporated into a phospholipid by monolayers of L-M fibroblasts. This phospholipid was identified as 1,2-diacyl-$\text{sn}$-glycero-3-phosphoisopropylethanolamine by using chemical and enzymatic procedures combined with thin-layer and gas-liquid chromatography. Since the phospho-N-isopropylethanolamine moiety is removed by phospholipase C, the stereochemistry of the phospholipid analog is identical to naturally occurring phosphoglycerides. Incubation of cells in 10 mM N-isopropylethanolamine inhibited the incorporation of [¹⁴C]choline and [¹⁴C]ethanolamine into phospholipids and stimulated the incorporation of [1-¹⁴C]palmitic acid and [1-¹⁴C]hexadecanol into triacylglycerols and alkyldiacylglycerols. These results indicate that N-isopropylethanolamine affects glycerolipid synthesis at the diradylglycerol branch point.     

Biosynthesis of Acyl Lipids Containing Very-Long Chain Fatty Acids in Microspore-Derived and Zygotic Embryos of Brassica napus L. cv Reston

Biosynthesis of very long chain (>C₁₈) fatty acids (VLCFAs) and the pathway for their incorporation into acyl lipids was studied in microspore-derived (MD) and zygotic embryos of Brassica napus L. cv Reston. In the presence of [1-¹⁴C]oleoyl-coenzyme A or [1-¹⁴C] eicosenoyl-coenzyme A, malonyl-coenzyme A, and reducing equivalents, maximal in vitro elongation activity was expressed in protein preparations from early-mid cotyledonary stage MD embryos (17-20 days in culture), when endogenous eicosenoic (20:1) and erucic (22:1) acids were just beginning to accumulate (approximately 1.5 milligrams per gram dry weight). The biosynthesis of VLCFAs and their incorporation into glycerolipids in vitro in the MD embryo system occurred at rates comparable to those measured in developing zygotic Reston embryos at about 20 days postanthesis. When glycerol-3-phosphate was supplied as acyl acceptor in time-course experiments using homogenates prepared from 18-day MD embryos, newly synthesized [¹⁴C]20:1 and [¹⁴C]22:1 were incorporated primarily into triacylglycerols (TAGs) and, to a lesser extent, into lyso-phosphatidic/phosphatidic acids, diacylglycerols, and phosphatidylcholines as well as the acyl-coenzyme A and free fatty acid pools. [¹⁴C]24:1 was not detected in any acyl lipid. Stereospecific analyses of the radiolabeled TAGs indicated that [¹⁴C]20:1 and [¹⁴C]22:1 moieties were esterified predominantly at the sn-3 position, but were also found at the sn-1 position. [¹⁴C]20:1, but not [¹⁴C]22:1, was detected at the sn-2 position. Similar patterns of ¹⁴C-labeled VLCFA distribution were obtained in experiments conducted using a 15,000g pellet fraction from 18-day MD embryos. All trends observed in the formation of TAGs containing VLCFAs in the Reston MD embryo system were also confirmed in studies of zygotic embryos of the same cultivar. The data support the biosynthesis of 20:1 and then 22:1 via successive condensations of malonyl-coenzyme A with oleoyl-coenzyme A and, for the first time in B. napus, demonstrate the incorporation of newly synthesized VLCFAs into TAGs via the Kennedy pathway.     

Evidence that the lipid carrier for N-acetylglucosamine is different from that for mannose in mung beans and cotton fibers

Cell-free enzyme particles from mung beans (Phaseolus aureus) or cotton (Gossypium hirsutum L.) fibers catalyze the incorporation of mannose from GDP-[¹⁴C]mannose and N-acetylglucosamine from UDP-[³H]-N-acetylglucosamine into polyprenyl-type lipids. These lipids have been synthesized and purified and the lipid moieties compared to each other as well as to dolichyl phosphate and to lipids isolated from similar mannoseand N-acetylglucosamine-containing lipids from liver and aorta.

The following lines of evidence indicate that in plants, the lipid carrier for N-acetylglucosamine is different from the lipid carrier for mannose: [List: see text]

We propose that the apparent difference in the lipid carrier for these two sugars may be a point of control of glycoprotein synthesis.

     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

Further investigations of the incorporation of [1-14C]acetate into the lipids of the silkworm Bombyx mori L

1. After the injection of sodium [1-¹⁴C]acetate, the highest incorporation of ¹⁴C into the lipids of the silkworm was observed after 24hr. 2. The specific radioactivity of the palmitic acid fraction was greater and increased more rapidly than that of the stearic acid fraction, which was consistent with the precursor–product relationship to be expected on the basis of current concepts of fatty acid synthesis in vivo. 3. The results indicate the probability of synthesis of lipid components in tissues other than the fat body. 4. Fractionation studies indicate considerable differences in the rate of incorporation of [1-¹⁴C]acetate into neutral lipids and phospholipids between larvae and pupae as well as among tissues of larvae. 5. The rate of incorporation of [1-¹⁴C]acetate remains constant throughout pupal development.     

Uptake of 13C-Tracer Arachidonate and γ-Linolenate by the Brain and Liver of the Suckling Rat Observed Using 13C-NMR

Arachidonic acid (AA; 20:4n-6) is one of the principal components of the phosphoglycerides in neural cell membranes. During the critical period of postnatal development in mammals, AA is supplied preformed, directly from the milk or derived from precursor fatty acids such as gamma-linolenic acid (GLA; 18:3n-6). In this study, 13C-NMR spectroscopy was applied to investigate the incorporation of [1-(13)C]AA and [3-(13)C]GLA into liver and brain lipids of 7-15-day-old rats. The main objective was to establish the importance of dietary GLA for tissue AA accretion relative to the contribution from preformed dietary AA. [1-(13)C]AA and [3-(13)C]GLA were injected into the stomach of 7-day-old rats as a mixture. 13C-NMR spectroscopy of lipid extracts revealed incorporation of [1-(13)C]AA and [5-(13)C]AA (the latter derived from metabolism of the injected [3-(13)C]GLA) into phosphoglycerides and triacylglycerols. Preformed AA was 10 (liver)-17 (brain) times more efficient in contributing to tissue AA than AA derived from precursor GLA. In separate experiments, NMR spectroscopy was used to assess uptake of [1-(13)C]AA directly in living rats and intact organs. Results showed that intact liver and brain contain an appreciable amount of NMR-detectable lipids. The in vivo/in vitro information obtained from organs provided details on the mobility and turnover of tissue lipids.     

Catabolism of myo-Inositol to Precursors Utilized for De Novo Glycerolipid Biosynthesis

Systemic injection of [2-3H]myo-inositol into frogs resulted in the incorporation of more than half of the label into glycerolipid classes other than phosphoinositides in retinal rod outer segment membranes. Following methanolysis and differential extraction of isolated lipid classes, radioactivity was recovered primarily in the aqueous phase. After phospholipase C hydrolysis of the total membrane lipids, 97% of the radioactivity was extractable with organic solvents, and 70% of the label in lipids was in 1,2-diglycerides. These results indicate that the label was incorporated primarily into the glyceryl moiety of the membrane glycerolipids. Intraocular injection of frog eyes or in vitro incubation of frog retinas with [2-3H]myo-inositol resulted in the incorporation of radioactivity almost exclusively into phosphoinositides in rod outer segment membranes. Incubation of retinas with [U-14C]glucuronic acid did not result in the formation of labeled retinal lipids. These results suggest that myo-inositol can be catabolized systemically to precursors utilized for glycerolipid biosynthesis in the retina.     

Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds

Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1-¹⁴C]oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and [2-¹⁴C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerois and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1-¹⁴C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2-¹⁴C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols.