Relative significance of exogenous and de novo synthesized fatty acids in the formation of rumen microbial lipids in vitro

Mixed rumen microorganisms (MRM) or suspensions of rumen Holotrich protozoa obtained from a sheep were incubated anaerobically with [1-(14)C]linoleic acid, [U-(14)C]glucose, or [1-(14)C]acetate. With MRM, the total amount of fatty acids present did not change after incubation. An increase in fatty acids esterified into sterolesters (SE) and polar lipids at the expense of free fatty acids was observed. This effect was intensified by the addition of fermentable carbohydrate to the incubations. Radioactivity from [1-(14)C]linoleic acid was incorporated into SE and polar lipids with both MRM and Holotrich protozoa. With MRM the order of incorporation of radioactivity was as follows: SE > phosphatidylethanolamine > phosphatidylcholine. With Holotrich protozoa, the order of incorporation was phosphatidylcholine > phosphatidylethanolamine > SE. With MRM the radioactivity remaining in the free fatty acids and that incorporated into SE was mainly associated with saturated fatty acids, but a considerable part of the radioactivity in the polar lipids was associated with dienoic fatty acids. This effect of hydrogenation prior to incorporation was also noted with Holotrich protozoa but to a much lesser extent. Small amounts of radioactivity from [U-(14)C]glucose and [1-(14)C]acetate were incorporated into rumen microbial lipids. With protozoa incubated with [U-(14)C]glucose, the major part of incorporated radioactivity was present in the glycerol moiety of the lipids. From the amounts of lipid classes present, their radioactivity, and fatty acid composition, estimates were made of the amounts of higher fatty acids directly incorporated into microbial lipids and the amounts synthesized de novo from glucose or acetate. It is concluded that the amounts directly incorporated may be greater than the amounts synthesized de novo.     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Hepatic lipase-mediated hydrolysis versus liver uptake of HDL phospholipids and triacylglycerols by the perfused rat liver

Hepatic triacylglycerol-lipase-mediated hydrolysis and liver uptake of high-density lipoprotein (HDL) lipid components were studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and active at the vascular bed. Human native HDL were labelled with tri-[3H]oleoylglycerol, [N-methyl-3H]dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl,2-[14C]linoleoylphosphatidylcholine (PLPC), 1-palmitoyl,2-[14C]linoleoylphosphatidyl-ethanolamine (PLPE) and 1-palmitoyl,2-[14C]palmitoylphosphatidylethanolamine (DPPE). (1) Relative degradation rates of phosphatidylethanolamine molecular species were 2- to 10-fold higher than those of phosphatidylcholine. Considering [14C] PLPC and [14C] PLPE as representative of HDL phosphatidylcholine and phosphatidylethanolamine, respectively, the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine generated after a 60 min perfusion were comparable. The enzyme showed a clear preference for the molecular species bearing an unsaturated fatty acid at the 2 position of glycerol; this was the most pronounced in the case of phosphatidylethanolamine molecular species. (2) Relative liver uptake of HDL-phosphatidylethanolamine was 4- to 5-fold higher than that of HDL-phosphatidylcholine, irrespective of the constitutive fatty acids. Nevertheless, mass estimation indicated that 3 times more molecules of phosphatidylcholine than of phosphatidylethanolamine were transferred. No correlation could be found between the relative degradation rates of phospholipids and their relative liver uptake, indicating a dissociation between the two processes. (3) Perfusate decay and relative liver uptake of labelled HDL-triacylglycerol were higher than that of any phospholipid class. No circulating radiolabelled free fatty acids accumulated in the perfusate, but they were found acylated into liver cell phospholipids and triacylglycerols. (4) A prior 10-12-min washout of the liver vascular bed with heparin removed over 80% of the hepatic lipase activity, as assessed by specific immunoinhibition. Hepatic lipase-depleted liver displayed impaired phospholipid hydrolysis and triacyglycerol uptake, whereas the transfer of HDL phospholipids to liver tissue was unaffected.     

Fatty acid and glycerol labeling of glycerolipids of leukocytes in response to ionophore A23187.

The effect of divalent cation ionophore, A23187, on the incorporation of [1-14C]palmitic acid, [1-14C]linoleic acid and [U-14C]glycerol into glycerolipids of polymorphonulcear leukocytes was examined. Ionophore A23187 stimulated the labeling of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and diacylglycerol by both labeled fatty acids and glycerol. [1-14C]Palmitic acid and [1-14C]linoleic acid incorporation into phosphatidylcholine and triacylglycerol was reduced by the presence of the ionophore in the incubation medium, while [U-14C]glycerol labeling of these lipids was not significantly changed under identical conditions. These data reflect that the acylation of sn-glycerol 3-phosphate is activated, and the acylations of lysophosphatidyl-choline and endogenous diacylglycerol are inhibited in cells incubated with ionophore A23187. External calcium was not required for the ionophore effect on the incorporation of labeled fatty acids and glycerol. It is suggested that the ionophore alters the metabolism of the fatty acid and glycerol moieties of glycerolipids by changing the distribution of intracellular calcium of leukocytes.     

Glycerolipid metabolism in lung from ventilated and unventilated rabbits

The incorporation of [14C]-glycerol 3-phosphate and [3H]-palmitate into phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and triacylglycerols by lung microsomes from ventilated and unventilated rabbits was measured. Unventilated lung microsomes showed an impairment of the "de novo" synthesis of phosphatidic acid and, therefore, a general decrease of glycerolipids synthesized from glycerol 3-phosphate. The incorporation of [3H]-palmitate into phosphatidic acid was considerably lower than the incorporation of [14C]-glycerol 3-phosphate by lung microsomes from both ventilated and unventilated rabbits, and the 3H/14C molar ratio did not change during incubation time. These observations suggest the preferential utilization of endogenous fatty acids by acyltransferases involved in the formation of phosphatidic acid. The activities of the enzymes implicated in the synthesis of phosphatidylcholine from lysophosphatidylcholine remained unchanged in lung from both ventilated and unventilated rabbits.     

Formation of omega- and (omega-1)-hydroxyfatty acids and plausible formation of ketofatty acid from microsomal phospholipids

Rat liver microsomes labeled with spin-labeled phosphatidylcholine release the label into the aqueous phase during the aerobic incubation with NADPH (Biochem. Biophys. Res. Commun. (1979) 87, 300-307). To establish the chemical nature of the released moiety, microsomes were labeled with [14C]phosphatidylcholine. When the 14C-labeled microsomes were incubated with NADPH under aerobic conditions, a few percent of the radioactivity was liberated into the aqueous phase within 60 min. Thin-layer chromatographic analysis of the radioactive substance liberated showed the presence of hydroxylated fatty acids derived from the 2-position of glycerol moiety. About one-third of the fatty acids formed from [14C]phosphatidylcholine during the incubation were converted into hydroxy-derivatives. Gas chromatography/mass spectrometry analysis further confirmed an NADPH-dependent formation of 16-hydroxypalmitic acid, 15-hydroxypalmitic acid, and hydroxy-derivatives of other fatty acids from the phospholipids of the microsomal membrane. Evidence was also obtained indicating the formation of ketopalmitic acid.     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

Effect of CO 2 concentration on phospholipid metabolism in the isolated perfused rat lung

Studies have been carried out on the incorporation of [U-(14)C]glucose, [2-(14)C]pyruvate, [2-(14)C]acetate, and [1-(14)C]-palmitate into the phospholipids of the isolated perfused rat lung in the presence of either 6 or 45 mm total CO(2) concentration in the perfusion medium. Incorporation of [U-(14)C]glucose into total phospholipid and into the phosphatidylcholine fraction was increased 19-53% over the 2-hr perfusion period in lungs perfused with medium containing 45 as compared with 6 mm CO(2). The incorporation of [2-(14)C]acetate, [2-(14)C]-pyruvate, and [1-(14)C]palmitate was not affected by the change in medium CO(2) concentration. Increased incorporation of [U-(14)C]glucose combined with a shift toward greater incorporation into the fatty acids of the phosphatidylcholine fraction produced a maximum increase of 90% in [U-(14)C]glucose incorporation into the fatty acids of phosphatidylcholine after 2 hr of perfusion in the presence of medium containing 45 mm CO(2) as compared with 6 mm CO(2). The increase in medium CO(2) concentration produced as much as a 150% increase in [U-(14)C]glucose incorporation into palmitate derived from the phosphatidylcholine fraction. The results provide evidence that glucose functions as an important precursor of palmitate in the phosphatidylcholine fraction of lung phospholipids and that the CO(2) concentration of the perfusion medium affects the incorporation of glucose into palmitate.     

Biosynthesis of triacylglycerols in the intestines of rainbow trout (Salmo gairdnerii) fed marine zooplankton rich in wax esters

Intestinal preparations from rainbow trout fed a diet rich in wax esters incorporated [1(-14)C]hexadecanoic acid and [1(-14)C]hexadecanol into triacylglycerols at the same rate. The ratio of the number of H atoms from C1 of hexadecanol to the number of molecules of hexadecanol incorporated into triacylglycerols was 1.6 : 3.0. [U-14C]Glucose was incorporated much faster into the glycerol moiety of triacylglycerols than was [U-14C]aspartic acid. We conclude that the oxidation of absorbed fatty alcohol to fatty acid and its subsequent incorporation into triacylglycerols is closely linked with the reductive formation of triacylglycerol-glycerol from glucose. The ability of trout intestines to metabolise fatty alcohol to triacylglycerols was the same in fish fed wax esters as in those fed triacylglycerols.     

Carbons from choline present in the phospholipids of Pseudomonas aeruginosa

The phospholipid composition of Pseudomonas aeruginosa grown in a mineral medium with choline as the carbon source was: phosphatidylethanolamine, 71.6±1.4%; phosphatidylglycerol, 11.8±0.4%; diphosphatidylglycerol, 0.8±0.4%; phosphatidic acid, 2.4±0.6%; lysophosphatidylethanolamine, 1.6±0.3%; phosphatidylcholine 7.9±0.3%; lysophosphatidylcholine, 3.9±0.7%. The molar ratio between the acidic and the neutral phospholipids was 0.18. Radiolabeling experiments with [methyl-¹⁴C]choline or [1,2-¹⁴C]choline carried out in cell suspension from bacteria that were grown in the presence of choline as the sole carbon source demonstrated that the carbons of the N-methyl groups of choline contributed to the synthesis of fatty acids while the carbons comprising the backbone of choline were used for the synthesis of glycerol.     

In vitro lipid metabolism in the rat pancreas. III. Effects of carbamylcholine and pancreozymin on the turnover of phosphatidylinositols, 1,2-diacylglycerols and phosphatidylcholines

1. The turnover of phosphatidylinositols and other glycerolipids was examined in rat pancreatic fragments incubated in the presence of carbamylcholine and pancreozymin used at a concentration inducing maximal alpha-amylase hypersecretion. 2. In stimulated tissue, [1-14C]acetate-labeled fatty acids were incorporated into phosphatidylinositols, 1,2-diacylglycerols, and phosphatidic acids in preference to phosphatidylcholines, phosphatidylethanolamines, triacylglycerols, monoacylglycerols, and free fatty acids. Variations in the percent distribution of 14C among fatty acids and in specific activity of individual fatty acids in each lipid class suggested that the secretagogues reduced selection of newly synthesized 1,2-diacylglycerols which occurred in the resting state before their incorporation into phosphatidylinositols. Secretagogues also promoted recycling of endogenous 1,2-diacylglycerols (produced from hydrolysis of unlabeled glycerolipids) for the biosynthesis of phosphatidylinositols. 3. Increased rate of incorporation of [1-14C]palmitate, [1-14C]linoleate, [1-14C]arachidonate and [1(3)(n)-3H]glycerol into phosphatidylinositols was detrimental to phosphatidylcholines. 4. The lipolytic effects of carbamylcholine and pancreozymin as illustrated by the release of 1,2-diacylglycerols and free fatty acids, were markedly inhibited in calcium-free medium enriched with 1 mM EGTA but increased turnover of phosphatidylinositols as determined from incorporation of radioactive precursors was only moderately affected.     

In vitro lipid metabolism in the rat pancreas. I. Basal lipid metabolism.

1. The in vitro basal lipid metabolism of rat pancreatic fragments was compared with that in adipose tissue fragments and liver slices. 2. [1-14C]Acetate added to the media was mostly incorporated into palmitic acid and to a lesser extent into oleic acid. In addition, pancreatic tissue exhibited a marked capacity for elongation of polyunsaturated fatty acids by [1-14C]acetate and resulting desaturation when compared to adipose tissue and liver. 3. Data obtained in the presence of [U-14C]glucose, [1-14C]palmitate and 3H20 indicate that acetyl-CoA derived from glucose and from beta-oxidation of fatty acids contributed to de novo lipogenesis. 4. Oxidation of [1-14C]palmitic acid was 9-13 times higher in the pancreas than in adipose tissue or liver when expressed on a wet weight basis. 5. The fatty acid moiety of pancreatic glycerolipids could be derived from de novo synthesis, fatty acids added to the medium, or from fatty acids formed from the hydrolysis of endogenous lipids. The glycerol moiety could be derived either from glucose, or directly from glycerol through participation of glycerol kinase.     

Incorporation into phospholipid classes and metabolism via desaturation and elongation of various 14C-labelled (n-3) and (n-6) polyunsaturated fatty acids in trout astrocytes in primary culture

The incorporation and metabolism of [1-14C]18:3(n-3), [1-14C]20:5(n-3), [1-14C]18:2(n-6), and [1-14C]20:4(n-6) were studied in primary cultures of trout brain astrocytes. There were no significant differences between the amounts of individual fatty acids incorporated into total lipid at 22 degrees C, with greater than 90% of all the fatty acids being incorporated into polar lipid classes. The distributions of 18:2(n-6), 18:3(n-3), and 20:5(n-3) in individual phospholipid classes at 22 degrees C were very similar, with 57-63 and 18-24% being incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Approximately equal amounts of 20:4(n-6), approximately 30% of the total, were incorporated into each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The metabolism of the (n-3) fatty acids to longer-chain and more unsaturated species was significantly greater than that of (n-6) acids, but delta 4-desaturase activity was very low. A culture temperature of 10 degrees C increased the incorporation of all the fatty acids into total lipid and that of C20 fatty acids into polar lipid. At 10 degrees C, the incorporation of C20 fatty acids into phosphatidylethanolamine and phosphatidylinositol was increased, and the incorporation into phosphatidylcholine and phosphatidylserine was decreased. The distribution of C18 fatty acids was unchanged at the lower temperature, as was the desaturation and elongation of all the polyunsaturated fatty acids incorporated.     

Hydrolysis of chylomicron arachidonate and linoleate ester bonds by lipoprotein lipase and hepatic lipase

Chylomicrons labeled with [3H]arachidonic and [14C]linoleic acid were incubated with bovine milk lipoprotein lipase or rat postheparin plasma, containing both lipoprotein lipase and hepatic lipase. During incubation with bovine lipoprotein lipase, [3H]arachidonic acid was released from chylomicron triacylglycerols at a slower rate than [14C]linoleic acid. Only small amounts of [14C]linoleic acid were found as 1,2(2,3)-diacylglycerols, whereas a transient accumulation as [14C]monoacylglycerols was observed. In contrast, significantly more [3H]arachidonic acid was found as 1,2(2,3)-diacylglycerols than as monoacylglycerols at all time intervals investigated. The initial pattern of triacylglycerol hydrolysis by postheparin plasma was similar to that of bovine lipoprotein lipase. However, in contrast to the results obtained with bovine lipoprotein lipase, little [3H]1,2(2,3)-diacylglycerol accumulated. The addition of antiserum to hepatic lipase increased the amount of 3H found in 1,2(2,3)-diacylglycerols and inhibited the formation of free [3H]arachidonic acid. The antiserum also caused a significant inhibition of the hydrolysis of [3H]-but not of [14C]triacylglycerol. With regard to chylomicron phospholipids, the rate of hydrolysis of [14C]linoleoyl phosphatidylcholine with milk lipoprotein lipase was twofold higher than that of the [3H]arachidonyl phosphatidylcholine. However, the hepatic lipase of postheparin plasma had similar activity towards the two phosphatidylcholine species. Postheparin plasma rapidly hydrolyzed chylomicron 3H-labeled and 14C-labeled phosphatidylethanolamine to the same degree, and lipoprotein lipase similarly hydrolyzed 3H-labeled and 14C-labeled phosphatidylethanolamine at approximately equal rates. Antiserum to hepatic lipase inhibited the postheparin plasma hydrolysis of phosphatidylethanolamine and 3H-labeled phosphatidylcholine by about 60%, but the 14C-labeled phosphatidylcholine by only 27%.(ABSTRACT TRUNCATED AT 250 WORDS)     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.     

The mechanism of intestinal absorption of phosphatidylcholine in rats

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with (32)P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([(14)C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([(14)C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of (32)P-labelled phosphatidylcholine or (32)P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and P(i) in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and P(i). The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway.     

Synthesis of fluorescent and radiolabeled analogues of phosphatidic acid

Procedures for the synthesis of fluorescent and radiolabeled analogues of phosphatidic acid are described. The fluorophore 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was coupled to 6-amino-caproic acid and 12-aminododecanoic acid by reaction of NBD-chloride with the amino acids under mild alkaline conditions at room temperature. 1,2-Dioleoyl-sn-[U-14C]glycerol 3-phosphate was prepared by acylation of sn-[U-14C]glycerol 3-phosphate with oleic acid anhydride using dimethylaminopyridine as the catalyst. This compound was converted to 1-oleoyl-sn-[U-14C]glycerol 3-phosphate by hydrolysis with phospholipase A2. The lysophosphatidic acid was reacylated with NBD-aminocaproyl imidazole or NBD-aminododecanoyl imidazole to form the fluorescent, radiolabeled analogue of phosphatidic acid. Fluorescent, non-radiolabeled analogues of phosphatidic acid were prepared by phospholipase D hydrolysis of fluorescent phosphatidylcholine.     

In vitro and in vivo synthesis of long-chain fatty acids from (1-14C) acetate in the renal papillae of rats.

1. The relationship between the rate of [1-14C] acetate incorporation into the fatty acids of renal papillary lipids and the acetate concentration in the medium has been measured. 2. [1-14C] acetate was incorporated mainly into fatty acids of phospholipids and triacylglycerols. Only a few per cent of the radioactivity was found in the free fatty acid fraction. 3. The major part of the [1-14C] acetate was found to be incorporated by a chain elongation of prevalent fatty acids. The major component of the poly-unsaturated fatty acids in triacylglycerols and the major product of fatty acid synthesis from [1-14C] acetate in vitro was demonstrated by mass spectrometry to be docosa-7,10,13,16-tetraenoic acid. 4. The radioactivity of docosa-7,10,13,16-tetraenoic acid accounted for 40% of total radioactivity in triacylglycerol fatty acids (lipid droplet fraction) and 20% of total radioactivity in membrane phospholipid fatty acids.     

Effects of 1,25-dihydroxyvitamin D-3 on phospholipid metabolism in chick myoblasts

1,25-Dihydroxyvitamin D-3 has been shown to increase phosphatidylcholine and decrease phosphatidylethanolamine levels of myoblasts. Recent studies have suggested that the metabolite stimulates the methylation of phosphatidylethanolamine into phosphatidylcholine. In addition, the sterol increases the arachidonate content of phosphatidylcholine. Experiments were carried out to identify the steps of muscle cell lipid metabolism affected by 1,25-dihydroxyvitamin D-3. Primary cultures of chick embryo myoblasts pretreated with physiological concentrations of 1,25-dihydroxyvitamin D-3 were labelled with [14C]ethanolamine. The sterol increased the incorporation of precursor into dimethylphosphatidylethanolamine and phosphatidylcholine, whereas it decreases the labelling of phosphatidylethanolamine. Prior treatment with cycloheximide and actinomycin D blocked these changes. 1,25-Dihydroxyvitamin D-3 also stimulated the incorporation of [14C]ethanolamine into CDP-ethanolamine. In addition, the sterol increased the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction but did not affect the incorporation of [14C]palmitic acid. The incorporation of labelled fatty acids into diacylglycerol was not changed by the sterol, whereas it stimulated incorporation of both precursors into triacylglycerol. The data indicate that 1,25-dihydroxyvitamin D-3 enhances the synthesis of phosphatidylcholine through a stimulation of de novo synthesis and methylation of phosphatidylethanolamine via a nuclear mechanism. The sterol may also increase the polyunsaturated fatty acid content of phosphatidylcholine by means of an activation of its deacylation-reacylation cycle.     

The effect of hypolipidemic drugs on plant lipid metabolism

The effect of hypolipidemic drugs, WY14643 and DH990, on plant lipid metabolism has been studied. The total incorporation of [14C]acetate into lipids was inhibited by addition of both drugs to aged potato (Solanum tuberosum) tuber discs, spinach (Spinacia oleracea) leaves, and spinach chloroplasts, while the incorporation in Chlorella vulgaris cells was affected only by DH990. Moreover, DH990 inhibited the incorporation of 14C-labeled fatty acids into phosphatidylcholine and phosphatidylethanolamine of potato discs, and decreased the incorporation into phosphatidylglycerol of Chlorella cells. DH990 inhibited the formation of polyunsaturated fatty acids in potato discs, Chlorella cells, and spinach leaves, whereas WY14643 had no effect on the formation of these fatty acids. Stearoyl-ACP desaturase from safflower (Carthamus tinctorius) seeds was very sensitive to both drugs, especially DH990, which completely blocked the activity at 2 mM levels. When safflower lysophospholipid acyltransferases were solubilized by detergent treatment, only DH990 inhibited the incorporation of [14C]oleoyl-CoA into lysophosphatidylcholine or lysophosphatidylethanolamine. Both drugs inhibited fatty acid synthesis from [14C]malonyl-CoA in the microsomal fraction from safflower seeds, but only DH990 inhibited FAS activity in the soluble fraction; both drugs inhibited severely the formation of stearic acid. Both acetyl-CoA carboxylase and acetyl-CoA synthetase were sensitive to both drugs.