Biosynthesis of liver glycerolipids from normal and essential fatty acid-deficient rats:3H-glycerol and 1-14C linoleic acid incorporation

Normal and essential fatty acid (EFA)-deficient rats were injected via the portal vein with a labeled solution containing³H-glycerol and 1-¹⁴C-linoleic acid during a 1 min period. Livers were immediately frozen, pulverized, and the lipids extracted and fractioned by thin layer chromatography. The incorporation of³H-glycerol and 1-¹⁴C-linoleic acid into the different lipid fractions was measured, and the per cent distribution and specific radioactivity determined. A parallel increase was found between the specific activity and the amount of³H-glycerol incorporated into 1,2-diglycerides, triglycerides, lecithin and cephalin from EFA-deficient and normal rats. Since the amount of glycerol in each fraction studied was quite similar in both groups of rats, these findings can explain the increase in the specific activity observed in the EFA-deficient rats. Nevertheless these facts do not necessarily imply an increased turnover rate of these molecules, since we do not know the specific radioactivity of the 1,2-diacylglycerol precursors. A remarkable increase in the specific radioactivity of the¹⁴C-linoleic acid incorporated into lipid fractions from EFA-deficient rats compared with control rats was observed. While the amount of 1-¹⁴C-linoleic acid incorporated into neutral lipids was similar in both groups of rats, a statistically significant increase in the amount of the label incorporated into phospholipids from EFA-deficient rats was observed. These facts suggest an increased turnover rate of the radiolinoleic acid into phospholipid molecules from EFA-deficient rats via deacylation-reacylation pathway.     

Lipogenesis in the developing brain from intracranially administered [1-14C] acetate and [U-14C] glucose

Fifteen-day-old rats divided into two groups were given [1-¹⁴C]acetate or [U-¹⁴C] glucose by intracranial injection and were sacrificed after 1 hr. Analysis of lipids from the two groups showed differences in the incorporation of radioactivity in the polar lipids and cholesterol. Analysis of brain fatty acid showed that whereas radioactivity from acetate was incorporated into saturated, monoand polyunsaturated fatty acids, the radioactivity from [U-¹⁴C] glucose was found only in 16∶0, 18∶0, and 18∶1. No radioactivity was found in polyunsaturated fatty acids even after concentration of this fraction by AgNO₃:SiO₂ thin layer chromatographic method. This difference is discussed in hypothetical terms of nonhomogeneous acetyl CoA pool, formation of acetyl CoA from glucose exclusively inside the mitochondria, and activation of injected acetate to acetyl CoA.     

The biosynthesis of cyclopropane and cyclopropene fatty acids in higher plants (Malvaceae)

The biosynthesis of cyclopropane and cyclopropene fatty acids has been investigated in immature seeds, leaves and callus tissue cultures of several species of Malvaceae. Chemical characterization of labeled cyclopropane and cyclopropene fatty acids obtained from incubations withl-[¹⁴CH₃] methionine confirmed that the ring methylene group was derived from the methyl group of methionine. The variation with time in the distribution of radioactivity in the products of incubations with [¹⁴CH₃] methionine and [2-¹⁴C] acetate suggested that the pathway involved initial formation of dihydrosterculic acid from oleic acid with subsequent desaturation to sterculic acid and α-oxidation to malvalic and dihydromalvalic acids. Direct evidence in favor of this pathway was provided by the conversion of [1-¹⁴C] oleic acid to dihydrosterculic and sterculic acids and by the desaturation of [1-¹⁴C] dihydrosterculic acid to sterculic acid, the first time that these processes have been demonstrated in higher plants. No conversion of [1-¹⁴C] stearolic acid to sterculic acid could be obtained under the same conditions. The presence of an active fatty acid α-oxidation system was demonstrated in the callus cultures.     

Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes

Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose,³H₂O, and¹⁴C-labeled amino acids (−)-Hydroxycitrate depressed the incorporation of³H₂O and [¹⁴C] alanine into fatty acids and cholesterol. Incorporation of [U-¹⁴C] leucine into lipids was not affected but incorporation of³H₂O into lipids was decreased significantly by (−)-hydroxycitrate. (−)-Hydroxycitrate depressed the incorporation of radioactivity from [2-¹⁴C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-³H]leucine 35 and 28%. As [2-¹⁴C]leucine labels the acetyl-CoA pool and [4,5-³H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.     

Metabolism of glyceryl 1-14C-trilinoleate in rat testis

Animals of the Sprague-Dawley strain were injected intratesticularly with radioactive glyceryl 1-¹⁴C-trilinoleate in a sequential experiment and killed at 1/4, 1/2, 1, 3, 6, 12, 24, 36 and 48 hr. Distribution and concentration (specific activity) of radioactivity among the lipid classes and fatty acids were determined. The results showed that radioactive 1-¹⁴C-linoleic acid was released from the glyceryl trilinoleate and incorporated throghout the lipid classes. The pattern of the distribution of the radioactivity and specific activities showed that the transformation of linoleic acid between the triglyceride, diglyceride and fatty acid pools was an equilibrium process. Linoleic acid released from glyceryl 1-¹⁴C-trilinoleate was converted to higher polyunsaturated fatty acids which were incorporated throughout the lipid classes, and was catabolized as evidenced by the finding of radioactivity in palmitic acid. The main metabolic pools in the interconversion of linoleic acid were arachidonic and 22∶5 acids. Small amounts of 20∶3 and 22∶4 were also detected and had high specific activities indicative of their roles as precursors.     

Metabolism of arachidonate and stearate injected simultaneously into the mouse brain

The metabolism of a polyunsaturated and a saturated fatty acid in brain membrane phosphoglycerides was examined by injecting simultaneously a mixture of¹⁴C-arachidonate and³H-stearate into the mouse brain and isolating the microsomal and synaptosomal fractions at 1–40 min after injections. Both types of labeled fatty acids were utilized more readily in the microsomal than the synaptosomal fractions in brain. However, labeled arachidonate was incorporated more rapidly into membrane phosphoglycerides than was stearate. In both subcellular fractions, the relative specific radioactivity (³H and¹⁴C) of diacyl-glycerophosphorylinositol (diacyl-GPI) was higher than other types of phosphoglycerides such as diacyl-glycerophosphorylcholine (diacyl-GPC) and diacyl-glycerophosphorylethanolamine (diacyl-GPE). Furthermore, the apparent rates of incorporation of radioactivity into diacyl-GPI was more rapid for the¹⁴C-arachidonate than for the³H-stearate. Results of the experiment have demonstrated obvious differences in metabolism between stearate and arachidonate in brain. The more rapid transfer of arachidonate to diacyl-GPI is probably due to the presence of an acyl transferase system specially active for the transfer of arachidonyl groups to diacyl-GPI.     

The incorporation of orally administered radiolabeled dihomo γ-linolenic acid (20∶3ω6) into rat tissue lipids and its conversion to arachidonic acid

Radioactivity from orally administered radiolabeled dihomo-γ-linolenic acid (20∶3ω6) was recovered from the liver, plasma and brain lipid fractions. After administration the fatty acid was metabolized to arachidonic acid, the 22 carbon chain length fatty acid, and was also β-oxidized. However, 22 hr after administration of [1-¹⁴C]20∶3 between one-third and one-half of the recovered radioactivity was still associated with dihomo-γ-linolenic acid in the liver and plasma lipid fractions. Orally administered dihomo-γ-linolenic acid is incorporated into lipid fractions and is, therefore, available in the metabolic pool for PGE₁ synthesis.     

Studies of differential turnover of palmitoyl and stearoyl species of glycerophosphatides using labeled unsaturated acids

Normal and bile fistula rats were injected with 1-¹⁴C-linoleate and arachidonate as albumin complex and the glycerides and glycerophosphatides of the liver and bile were isolated at various time intervals. The distribution of radioactivity among the individual molecular species was determined by thin layer and radio gas chromatography and specific enzymic hydrolyses. At 30 min after administration of linoleate 30% of the radioactivity in liver was in lecithins and 8% in cephalins, while at 120 min 48% was in lecithins and 16% in cephalins. After arachidonate, 58% and 64% of the counts were in lecithins and 12% to 13% in the cephalins at the above periods of sampling. The specific activity of the palmitoyl linoleoyl lecithins and cephalins was two to three times higher than that of the corresponding stearoyl linoleoyl species, which was of the same order but much lower magnitude than found previously for lecithins using labeled phosphate and choline. The palmitoyl and stearoyl species of arachidonoyl lecithins possessed equal specific activities, in sharp contrast to previous findings with radioactive phosphate, which showed a 12 times higher specific activity for the palmitoyl arachidonate. The palmitoyl arachidonoyl cephalins had two to three times greater specific activity than the corresponding stearoyl species in agreement with previous work using labeled phosphate. The distribution of radioactivity suggests that the arachidonate was incorporated into the lecithins largely via acyl transfer, while the linoleate contributed to both acyl transfer and de novo synthesis. Interpretation of the mechanism of uptake of these acids into the cephalins awaits further studies. Presented in part at the Federation of American Societies for Experimental Biology Meeting, Atlantic City, April 1970.     

Incorporation of radioactive polyunsaturated fatty acids into liver and brain of developing rat

The incorporation of radioactivity from orally administered linoleic acid-1-¹⁴C, linolenic acid-1-¹⁴C, arachidonic acid-³Hg, and docosahexaenoic acid-¹⁴C into the liver and brain lipids of suckling rats was studied. In both tissues, 22 hr after dosing, 2 distinct levels of incorporation were observed: a low uptake (from 18∶2-1-¹⁴C and 18∶3-1-¹⁴C) and a high uptake (from 20∶4-³H₈ and 22∶6-¹⁴C). In adult rats, the incorporation of radioactivity into brain lipids from 18∶2-1-¹⁴C and 20∶4-³H was considerably lower than the incorporation into the brains of the young rats. In the livers of the suckling rats, the activity from the 18 carbon acids was associated mostly with the triglyceride fraction, whereas the activity from the 20∶4-³H₈ and 22∶6-¹⁴C was concentrated in the phospholipid fraction. In the brain lipids, the activity from the different fatty acids was associated predominantly with the phospholipids. In the liver and brain phospholipid fatty acids, some of the activity in the 18∶2-1-¹⁴C and 18∶3-1-¹⁴C experiments was associated with 20 and 22 carbon polyunsaturated fatty acids; however, radioactivity from orally administered 20∶4-³H₈ and 22∶6-¹⁴C was incorporated intact into the tissue phospholipid to a much greater extent compared with the incorporation of radioactivity into 20∶4 and 22∶6 in the experiments where 18∶2-1-¹⁴C and 18∶3-1-¹⁴C, respectively, were administered. Possible reasons for these differences are discussed. Rat milk contains a wide spectrum of polyunsaturated fatty acids, including linoleate, linolenate, arachidonate, and docosahexaenoate. During the suckling period in the rat, there is a rapid deposition of 20∶4 and 22∶6 in the brain. The results of the present experiments suggested that dietary 20∶4 and 22∶6 were important sources of brain 20∶4 and 22∶6 in the developing rat.     

Fatty acid biosynthesis and incorporation into lipid classes in seeds and seed tissues of flax

Fatty acid metabolism in developing flaxseeds was studied by incubating whole seeds or isolated seed tissues in buffered solutions of 1-¹⁴C-acetate, 2-¹⁴C-malonate and¹⁴CO₂. Lipid classes were separated by thin layer chromatography, and fatty acid labeling in phospholipids, diglycerides and triglycerides was determined by combined thin layer and gas liquid chromatographic techniques. Incorporation of¹⁴C from acetate into embryo lipids was very rapid with phospholipids and 1,2-diglycerides becoming highly labeled in treatment times as short as 5 min. Triglycerides were labeled more slowly. Phospholipid radioactivity was largely associated with the phosphatidyl choline fraction. Oleic acid had the highest specific activity of all major fatty acids in short treatment periods. This was followed in decreasing order of activity by palmitic, linoleic, stearic and linolenic acids. As the treatment period was lengthened to 90 min or longer, linoleic and linolenic activities were markedly increased. Use of malonate or CO₂ rather than acetate as the substrate increased the labeling of the saturated acids. Incorporation of¹⁴C from acetate into lipids of endosperm tissues and whole flax seeds was slower than incorporation into embryo lipids. Stearate had the highest specific activity of the fatty acids in endosperm and whole seeds. Presented in part at the AOCS Meeting in New York, October 1968.     

Absorption and metabolism of 1-14C-methyl linoleate hydroperoxide

The absorption and metabolism of 1-¹⁴C-methyl linoleate hydroperoxide by rats was investigated. After intubation with 2 mg of peroxide, peak¹⁴CO₂ production occurred at 90 min and 25% of the dose was expired in 24 hr. Fortyfive per cent stil remained in the gastrointestinal tract after 24 hr, most of it bound to the stomach epithelium in the form of intact peroxide. Lymph was collected from the thoracic duct 2 hr after intubation and examined for labeled metabolites. Seven per cent of the radioactivity in the lymph was present in a free 1-¹⁴C-hydroxy fatty acid and 31% in its methyl ester. Fifty-seven per cent occurred in lymph triglycerides where it was equally distributed between a 1-¹⁴C-trienoic fatty acid and an unidentified 1-¹⁴C-oxy acid. The radioactivity in liver lipids was associated mainly with randomly labeled normal fatty acids. No¹⁴C-hydroxy acids were detected in liver lipids and no evidence was obtained for the absorption of unchanged peroxide. The hydroxy and trienoic acids appear to be formed during absorption by a reduction-dehydration reaction sequence.     

Early steps on the in vivo incorporation of 1-14C-linoleic acid into liver lipids from normal and essential fatty acid deficient rats

Essential fatty acid (EFA) deficient rats were injected intraportally with a solution of 1-¹⁴C-linoleic acid during a 1 min period. Livers were quickly frozen, pulverized, and the lipids extracted and fractioned by thin layer chromatography. The incorporation of 1-¹⁴C-linoleic acid into liver lipids was measured. The results were compared with those previously obtained from normal rats. No significant differences were observed in the total radioactivity recovered from lipid extracts. While the distribution of radioactivity into the 1–2 diacylglycerol fraction remained unchanged in both groups of rats, in the EFA deficient rats the 1-¹⁴C-linoleic acid incorporation was actually directed to the phospholipid fractions instead of to the triacylglycerol fractions as was observed in the normal rats.     

Die Rolle von Prostaglandinen bei der hormonal stimulierten Lipolyse in isolierten Fettgeweben

The Role of Prostaglandins in Hormone-Stimulated Lipolysis in Isolated Fatty Tissues Biosynthesis of PGE from ¹⁴C labelled unsaturated fatty acids by enzyme preparations from epididymal fatty tissues of rats and rams is reported. Di-homo-γ-linolenic acid and arachidonic acid were detected in di- and triglyceride fractions of the fatty tissues of rats. The amounts found are perhaps sufficient for explaining quantitatively the synthesis of PGE in intact fatty tissues. The specificity of anti-lipolytic action of PGE homologues was studied. Prostaglandins from EFA-active fatty acids with 20 and 21 C-atoms are as active as PGE₁, whereas prostaglandins from EFA-inactive fatty acids are much less active. The possible action of PGE₁ on the formation and degradation of cyclic adenosine-3′,5′-monophosphate was studied with isolated enzyme system. A direct action of PGE₁ could not be detected.     

Effects of purified eicosapentaenoic acid on arachidonic acid metabolism in cultured murine aortic smooth muscle cells,vessel walls and platelets

The effects of highly purified eicosapentaenoic acid (97% pure) on the arachidonic acid cascade in isolated murine vascular cells and platelets were studied. The incorporation of eicosapentaenoic acid was not as active as that of arachidonic acid in platelets. The ratio of incorporation of eicosapentaenoic acid to arachidonic acid into platelet phospholipids was about 0.7. Analysis of the phospholipid fractions of platelets after labeling with¹⁴C-eicosapentaenoic acid and¹⁴C-arachidonic acid revealed that the incorporation of¹⁴C-eicosapentaenoic acid into the phosphatidylinositol fraction is significantly less than that of¹⁴C-arachidonic acid, while the incorporation of both fatty acids into other phospholipid fractions was almost the same. On the other hand, no significant difference between either fatty acid in incorporation rate, kinetics or distribution in cellular phospholipids was found in cultured aortic smooth muscle cells. Following treatment with eicosapentaenoic acid, cells produced less prostacyclin from endogenous arachidonic acid than did control cells. This was not due to the decrease in fatty acid cyclooxygenase activity, but rather, due to the decrease in arachidonic acid content in cellular phospholipids. In addition, eicosapentaenoic acid was neither converted to prostaglandin I₃ by the vascular cells nor to thromboxane A₃ by platelets. Furthermore, similar results were also obtained by in vivo experiments in which rats were fed with eicosapentaenoic acid enriched diet.     

Metabolism of 1-14C-methyl linoleate hydroperoxide in the rabbit

The metabolism of 1-¹⁴C-methyl linoleate hydroperoxide (1-¹⁴C-MLHP) by the rabbit was investigated. Administration of 1.1–1.9 mg of 1-¹⁴C-MLHP by ear vein injection proved lethal to four of the nine experimental animals. After 2 hr the lungs and liver contained 3.3% and 7.2%, respectively, of the dose. This radioactivity was found to be associated primarily with intact 1-¹⁴C-MLHP. The triglycerides from these tissues also contained¹⁴C-trienoic and¹⁴C-dienoic fatty acids. Of the dose, 68% was recovered as¹⁴CO₂ in 2 hr compared to 39% after 1-¹⁴C-methyl linoleate injection. The triglycerides from kidney adipose tissue contained a small amount of¹⁴C-hydroxy fatty acid, providing confirmation of previous evidence for the presence of a fatty acid hydroperoxide reductase in animal tissues.     

Absorption and metabolism of 1-14C-hydroxy octadecadienoate in the rat

The metabolism of 1-¹⁴C-9(13)-hydroxy octadecadienoic acid methyl ester (1-¹⁴C-HAME) by the rat was investigated in vivo and in liver slices. A 1.5 mg dose of 1-¹⁴C-HAME administered by stomach tube was efficiently hydrolyzed and absorbed from the intestinal tract. In comparison with 1-¹⁴C-methyl linoleate (1-¹⁴C-ML), 1-¹⁴C-HMAE was more extensively oxidized to¹⁴CO₂ in vivo and in vitro. After 1-¹⁴C-HAME administration as much as 50% of the radioactivity in the adipose tissue triglycerides was associated with¹⁴C-hydroxy fatty acids. The remaining activity was present in randomly labeled normal fatty acids. No evidence was obtained for the incorporation of¹⁴C-hydroxy acids into liver lipids; most of the radioactivity from 1-¹⁴C-HAME in this organ was recovered in saturated and monoenoic fatty acids. About 10% of the radioactivity 24 hr after 1-¹⁴C-HAME administration was associated with triglyceride trienoic acids, indicating that at least a portion of this acid was dehydrated in the liver. An unidentified polar acid was detected in the urine of the 1-¹⁴C-HAME-treated animals.     

Analysis of lipids containing hydroxy fatty acids

Lipids containing hydroxy fatty acids or hydroxyacyl moieties are acetylated with [1-¹⁴C]acetic anhydride or [³H]acetic anhydride, and the content of hydroxy fatty acids or hydroxyacyl moieties is estimated from the specific radioactivity of the acetylated products with respect to that of radioactive standards, such as radioacetylated ricinoleic acid or triricinoleoylglycerol. Mixtures of triacylglycerols containing one, two and three hydroxyl groups per molecule are derivatized in a similar manner, and the resulting acetates are fractionated by thin layer chromatography according to the number of acetate groups per molecule. The relative proportion of each type of triacylglycerols in the mixture is estimated from the distribution of radioactivity in the various fractions. Applications of these techniques are demonstrated by the analysis of several seed lipids.     

The in vivo incorporation of labeled linoleic acid, α-linolenic acid and arachidonic acid into rat liver lipids

The incorporation of 1-¹⁴C-linoleic acid, 1-¹⁴C-α-linolenic acid and 1-¹⁴C-arachidonic acid into rat liver lipids was measured and the per cent distribution of radioactivity into the different lipid fractions determined. Normal rats were injected into the portal vein with the labeled solutions during a one minute period. Livers were quickly frozen, pulverized, and the lipids extracted and fractioned by thin layer chromatography. No significant differences were observed in the amounts of labeled fatty acids incorporated per gram of rat liver. While 1-¹⁴C-linoleic acid and 1-¹⁴C-α-linolenic acid were found in appreciable amounts in the 1,2 diacylglycerol fraction, about one fifth as much 1-¹⁴C-arachidonic acid was esterified in this fraction. 1-¹⁴C-arachidonic acid was the leading acid esterified in the phospholipid fractions.     

In vivo incorporation of3H arachidonic acid and14C linoleic acid into liver lipids from essential fatty acid-deficient rats

Essential fatty acid (EFA)-deficient rats were injected intraportally with a labeled solution containing³H arachidonic acid and¹⁴C-linoleic acid during a 1 min period. Livers were quickly frozen, pulverized, and the lipids extracted and fractioned by thin layer chromatography. The incorporation of³H and¹⁴C into liver lipids was measured, and the per cent distribution of radioactivity into the different lipid fractions determined and compared with those previously obtained from normal rats. In contrast with normal rats, ca. 70% of the³H arachidonic acid and¹⁴C-linoleic acid incorporated into total lipids from EFA-deficient rats was recovered in the phospholipid fraction. From the results of this experiment, it is suggested that a more active deacylation-reacylation cycle in EFA-deficiency could be responsible for this increase.     

Polyenoic acid metabolism in cultured human skin fibroblasts

The incorporation of [1-¹⁴C]linoleic acid, and [1-¹⁴C]linoleic acid into cellular lipids of cultured human skin fibroblasts was studied. Cultured cells took up both labeled fatty acids at nearly the same rate and incorporated them into a variety of lipid classes. At the end of 1 hr incubation with [1-¹⁴C]linoleic acid, radioactivity was found in the triacylglycerol (TG) and choline phosphoglyceride (CPG) pools preferentially. Incorporation into the TG fraction decreased rapidly, while the uptake into CPG, serine phosphoglyceride (SPG), and ethanolamine phosphoglyceride (EPG) fractions increased progressively with longer incubation times. Similar results were obtained with [1-¹⁴C]linoleic acid as precursor. At the end of 24 hr, desaturation and chain elongation of 18∶3 n−3 was more extensive than conversion of 18∶2 n−6 to higher polyenoic acids. During pulse-chase experiments with either fatty acid precursor, the incorporated radioactivity was progressively lost from cellular lipids, particularly from the TG and CPG fractions, but continued to increase in the SPG and EPG pools. The similar labeling pattern of cellular phospholipids with linoleic or linolenic acids, and data from pulse-chase studies suggest that a direct transfer of fatty acids from CPG to EPG is a likely pathway in fibroblast cultures. Incorporation into the EPG pool during the pulse-chase experiments paralleled extensive desaturation and elongation of linoleic acid into 20∶4 n−6, and 22∶4 n−6; and of linolenic acid into 22∶5 n−3 and 22∶6 n−3.