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.     

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.     

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.     

Turnover of fatty acids in rat liver cardiolipin: Comparison with other mitochondrial phospholipids

Following intraperitoneal administration of 1-¹⁴C-linoleic acid or 2-³H-acetate to rats, the specific radioactivities of both liver cardiolipin and other mitochondrial phospholipids after different time intervals were measured. Comparison of the data obtained with those from another stock of rats treated with³²P-phosphate or 2-³H-glycerol showed that the fatty acids of cardiolipin, like those of other phospholipids, exhibit an independent turnover with respect to the remaining parts of the molecule. The half-life of acyl moieties of cardiolipin is ca. 20% higher than that of the same components of other mitochondrial phospholipids. Moreover, it appears that, in both cardiolipin and other phospholipids, linoleyl residues turn over faster than nonessential fatty acids. Discussion is made as to whether this characteristic can be related to the role of phospholipids in the functioning of some enzymes bound to the inner mitochondrial membrane.     

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.     

Inhibition of desaturation of stearic acid in livers of rats fed ethionine

The effect of ethionine on the conversion of stearic acid to oleic acid was studied. Rats were fed essential fatty acid (EFA) deficient diet for three weeks, after which time half the animals were fed 0.25% DL-ethionine for nine additional days. Seventeen hours prior to killing, they were fed a slurry of the diet containing 18-¹⁴C-stearic acid. Liver triglycerides and phospholipids were extracted and separated and their fatty acid composition and the distribution of radioactivity between stearic and oleic acid was determined. In the tissues studied, oleic acid was maintained at control levels in ethionine-fed rats, but eicosatrienoic acid was significantly depressed. Distribution of radioactivity and specific activity of oleic acid in the triglycerides and phospholipids were significantly reduced by the analogue. In vitro studies of desaturation and chain elongation reactions, with liver microsomes, using 18-¹⁴C-stearic and 1-¹⁴C-linoleic acids as substrates, showed that ethionine depressed the synthesis of oleic acid from stearic and γ-linolenic from linoleic acid. Elongation of linoleic adie to a 20∶2 fatty acid was unaffected by ethionine. Therefore, the results showed that ethionine inhibited desaturation of stearic to oleic acid in vivo and in vitro and probably also impaired the desaturation of oleic to octadeca-6, 9-dienoic acid. Maintenance of control levels of oleic acid in the tissues of ethionine-fed, EFA deficient rats suggested the presence of synthetic pathways for oleic acid other than via desaturation of stearic acid. Presented in part at the AOCS Meeting, San Francisco, April 1969.     

Effect of ATP on the microsomal desaturation of unsaturated fatty acids

The effect of ATP on the microsomal desaturation of linoleic acid to γ-linolenic acid was studied in a system in vitro with the following results: (1) preincubation of rat liver microsomes with ATP alone in N₂ or in the presence of CoA and Mg⁺⁺ followed by subsequent incubation with 1-¹⁴C-linoleic acid plus NADH in O₂ resulted in enhancement of 1-¹⁴C-linoleic acid desaturation when compared with control samples in which no preincubation was performed; (2) the preincubation of the microsomes with ATP, Mg⁺⁺ and CoA in the presence of 1-¹⁴C-linoleic acid decreased the desaturation of the labeled acid to γ-linolenic acid upon subsequent incubation with NADH, as a consequence of incorporation of the acid into the microsomal lipids; (3) the increase of linoleic acid desaturation depended on the ATP concentration during preincubation and followed a sigmoidal curve. It was specific for ATP, and neither GTP, CTP, ADP nor AMP produced a similar effec. However, GTP or CTP could replace ATP as a cofactor in the microsomal desaturation of free linoleic acid to γ-linolenic, suggesting that directly or indirectly they may activate conversion of the free acid to linoleyl-CoA; (4) preincubation of microsomes with ATP activated the acylation of CoA. However, this activation showed no quantitative correlation with enhancement of the desaturation reaction; (5) addition of ATP also stimulated conversion of linoleyl-CoA to γ-linolenic acid. This enhancement was not related to inhibition of the linoleyl-CoA hydrolase; (6) however, in spite of these results, preincubation with ATP did not increase the initial velocity of linoleic acid or linoleyl-CoA desaturation; (7) preincubation of microsomes with ATP also increased the 6-desaturation of oleic acid and α-linolenic acid but did not increase the 9-desaturation of plamitic and stearic acid.     

Tissue culture of cocoa beans (Theobroma cacao L.): Incorporation of acetate and laurate into lipids of cultured cells

Suspension cultures of cocoa bean tissue readily incorporated exogenous acetate into lipids. The distribution of radioactivity from acetate in individual lipid classes after 48 hr was 20, 5, 1, 15, 25, and 35% in triglycerides, diglycerides, free fatty acids, sterol esters, sterols and polar lipids, respectively. The labeled acetate was rapidly incorporated into various fatty acids within 2 hr. The [1-¹⁴C] saturated fatty acids declined slightly after 4 hr, whereas [1-¹⁴C] oleate declined significantly after 2 hr. There was a concomitant increase in [1-¹⁴C] linoleate. The radioactivity associated with linolenate was relatively high up to 4 hr, declined by 24 hr, and then increased again. The kinetics of fatty acid labeling suggested that biosynthesis of linolenic acid in cocoa bean suspension culture may occur via the desaturation of linoleic acid and the chain elongation of dodecatrienoic acid. The patterns of fatty acid radiolabeling following incubation of cells with [1-¹⁴C] laurate was consistent with this mechanism.     

Formation of diglycerides of long turnover time from labeled acetate and glucose in rat tissues

Rat adipose tissue pieces were incubated with acetate-2-¹⁴C and glucose-¹⁴C(U), respectively, and liver slices with acetate-2-¹⁴C. The labeled tissues were then reincubated in inactive medium, and the changes of radioactivity in the different lipid classes were determined. In all three experiments a significant amount of radioactivity was incorporated in the diglycerides. During 1 hr of reincubation in inactive medium the radioactivity of diglycerides decreased from 35 to 26% of the total lipid activity in the adipose tissue labeled with acetate. In the adipose tissue labeled with glucose radioactivity fell from 25 to 19%. In liver slices 11% of the labeled acetate was incorporated in the diglycerides, and during the 2 hr of reincubation this value fell to its half. The radioactivity of the uniformly labeled glucose was distributed equally in the fatty acids and the glycerol. The distribution of radioactive glycerol between diglycerides and triglycerides was similar to that of the labeled fatty acids. Triglyceride synthesis seems to always be accompanied by the formation of diglycerides with a lastint turnover time.     

Compartmental study of rat renal phospholipid metabolism

Phospholipid content and metabolism were studied in rat renal papillary, medullary and cortical slices. The highest concentration of phospholipids was found in cortex and the lowest in papilla samples (ratio cortex/medulla, 1.3; cortex/papilla, 3.7). The profile of the various phospholipids was different depending on the zone. The most important difference was the relative concentrations of sphingomyelin (CerPCho) and phosphatidylinositol (PtdIns) with ratios for PtdIns/CerPCho of 5.0, 3.3 and 2.5 in papilla, medulla, and cortex, respectively. In the three zones, PtdIns showed the highest specific activity for [2-¹⁴C]glycerol and [1-¹⁴C]arachidonic acid incorporation. By contrast, a higher amount of [1-¹⁴C]palmitic acid was incorporated into phosphatidylcholine than into any other phospholipid. The various radioactive precursors were only poorly incorporated into phosphatidylethanolamine. No radioactivity was associated with phosphatidylserine. The papilla possesses the most active phospholipid metabolism of all the pathways studied.     

Placental transport oftrans fatty acids in the rat

Placental transport of 9-trans [1-¹⁴C] octadecenoic (elaidic) and 9-trans,12-trans [1-¹⁴C] octadecadienoic (linoelaidic) acids was demonstrated in rats. On the 18th day of gestation, a¹⁴C-labeled albumin complex of elaidic or linoelaidic acid was injected into the jugular vein of pregnant rats. For comparison, 9-cis [1-¹⁴C] octadecenoic (oleic) or 9-cis,12-cis [1-¹⁴C] octadecadienoic (linoleic) acid also was injected into the maternal circulation of rats. All animals were sacrificed 1 hr following injection. Lipid composition and distribution of label were determined in maternal plasma, placental and fetal tissues. Differences in specific activities of plasma, placental and fetal total lipids indicated a decreasing concentration gradient for bothcis andtrans isomers of octadecenoic and octadecadienoic acids. Distribution of radioactivity in various lipid components was determined by thin layer chromatography. Irrespective of the label, the highest percentage of total radioactivity was carried by triglycerides (TG) in maternal plasma (∼60–80%), and was incorporated mainly in phospholipids (PL) of fetal tissues (∼50–60%). A nearly equal distribution of the label was found between PL and TG of placental lipids (∼40%). Radioactivity of fatty acid methyl esters (FAME) determined by radiogas liquid chromatography indicated that after injection of linoelaidate, radioactivity of maternal plasma, placental and fetal tissue FAME was associated only witht,t-18∶2. Following injection of elaidate, all the radioactivity in placental FAME was associated witht-18∶1; however, in fetal tissues, the label was distributed between 16∶0 andt-18∶1. These findings suggest that, in contrast to linoelaidic acid, rat fetal tissues can metabolize elaidic acid via β oxidation to form acetyl CoA and palmitic acid.     

Chain length specificity in the utilization of long chain alcohols for ether lipid biosynthesis in rat brain

A mixture ofcis-9-[1-¹⁴C] octadecenol and [1-¹⁴C] docosanol was injected into the brains of 19-day-old rats, and incorporation of radioactivity into brain lipids was determined after 3, 12, and 24 hr. Both alcohols were metabolized by the brain but at different rates; each was oxidized to the corresponding fatty acid, but oleic acid was more radily incorporated into polar lipids. Substantial amounts of radioactivity were incorporated into 18∶1 alkyl and alk-1-enyl moieties of the ethanolamine phosphoglycerides and into 18∶1 alkyl moieties of the choline phosphoglycerides. Even after the disappearance of the 18∶1 alcohol from the substrate mixture (12 hr), the 22∶0 alcohol was not used to any measurable extent for alkyl and alk-1-enyl glycerol formation.     

Incorporation of 1,2-14C-ethanolamine into subfractions of rat liver phosphatidylethanolamines and phosphatidylcholines

The incorporation of 1,2-¹⁴C-ethanolamine into the liver phosphatidylethanolamines (PE) and phosphatidylcholines (PC) in female rats was studied. These phosphatides were fractionated according to their degrees of unsaturation and the specific activities of monoenoic, dienoic, tetraenoic and hexaenoic fractions were measured at intervals of 1, 20, 60 and 300 min after injection of the labeled precursor. Hexaenoic and dienoic PE incorporated and lost the label rapidly. Although the labeled precursor was incorporated into tetraene PE at a similar rate, this fraction attained a relatively low specific activity that remained essentially constant between 10 and 300 min after injection of the label. Hexaenoic PC had the highest specific activity among the PC fractions at all time periods. Estimations of the rate of loss of radioactivity in the hexaenoic PE fraction and its appearance in hexaenoic PC indicate that the docosahexaenoic acid is conserved, possibly by being reincorporated into PE after becoming a part of the hexaenoic PC species. The high rate of turnover of the hexaenoic PE also suggests that this fraction might have some special role in endogenous choline synthesis.     

1-14C-Linoleic acid distribution in various tissue lipids of guinea pigs following an oral dose

A recent study on the metabolism of 1-¹⁴C-α-linolenic acid in the guinea pig revealed that the fur had the highest specific activity of all tissues examined, 48 h after dosing. The present study investigated the pattern of tissue lipid labeling following an oral dose of 1-¹⁴C-linoleic acid after the animals had been dosed for the same time as above. Guinea pigs were fed one of two diets with a constant linoleic acid content (18% total fatty acids) and a different content of α-linolenic acid (0.3 or 17.3%) from weaning for 3 wk and 1-¹⁴C-linoleic acid was given orally to each animal for 48 h prior to sacrifice. The most highly labeled tissues (dpm/mg of linoleic acid) were liver, followed by brain, lung and spleen, heart, kidney and adrenal and intestines, in both diet groups. The liver had almost a three-fold higher specific activity than skin and fur which was more extensively labeled than the adipose and carcass. Approximately two-thirds of the label in skin plus fur was found in the fur which, because of a low lipid mass, would indicate that the fur was highly labeled. All tissues derived from animals on the diet with the low α-linolenic acid level were significantly more labeled than the tissues from the animals on the high α-linolenic acid diet, by a factor of 1.5 to 3. The phospholipid fraction was the most highly labeled fraction in the liver, free fatty acids were the most labeled fraction in skin & fur, while triacyglycerols were the most labeled in the carcass and adipose tissue. In these tissues, more than 90% of the radioactivity was found in fatty acids with 2-double bonds in the tissue lipids. These data indicate that the majority of label found in guinea pig tissues 48 h after dosing was still associated with a fatty acid fraction with 2-double bonds, which suggests there was little metabolism of linoleic acid to more highly unsaturated fatty acids in this time frame. In this study, the labeling of guinea pig tissues with linoleic acid, 48 h after dosing, was quite different from the labeling with α-linolenic acid reported previously. The retention of the administered radioactivity from ¹⁴C-linoleic acid in the whole body lipids was 1.6 times higher in the group fed the low α-linolenic acid diet (diet contained a total of 1.8 g PUFA/100 g diet)compared with the group fed the high α-linolenic acid diet (diet contained 3.6 g PUFA/100 g diet). The lack of retention of ¹⁴C-labeled lipids in the whole body would be consistent with an increased rate of β-oxidation of the labeled fatty acid on the diet rich in PUFA, a result supported by other studies using direct measurement of labeled carbon dioxide.     

Metabolism of lipids in rat testes: Interconversions and incorporation of linoleic acid into lipid classes

Studies are reported on the mode of incorporation of linoleic acid into lipid classes of testicular lipids. 1-¹⁴C-linoleic acid was injected into the testes of adult rats of the Sprague-Dawley strain. Groups of animals were killed at 1, 3, 6, 12, 24 and 48 hr after injections of the radioactive linoleic acid. The testes of each animal and livers of some animals were excised. Fatty acid and lipid class comkposition of the extracted lipids of the testes of each animal were determined as well as the distribution of radioactvity in these compounds. Radioactive linoleic acid and fatty acids derived from it by interconversion and catabolism were incorporated into all the lipid classes. Incorporation of linoleic acid into the lipid classes was much faster than its interconversion or catabolism to other fatty acids. The importance of the fatty acid pool in the mode of incorporation of the fatty acids into the lipid classes is demonstrated.     

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.     

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.     

Metabolism of gossypol,biosynthesized from methyl-14C-and carboxyl-14C-labeled sodium acetate,in rat

Stereospecifically labeled radioactive gossypol was biosynthesized by incubating cotton seedlings with either methyl-¹⁴C- or carboxyl-¹⁴C-labeled sodium acetate. The respective products were purified as gossypol acetic acid. Each radioactive gossypol acetic acid preparation was dissolved in oil and administered by stomach tube to two rats. A negligible amount of radioactivity was found in the expired air of the rats receiving the gossypol biosynthesized from 1-¹⁴C-sodium acetate; however, a significant quantity of radioactivity was found in the expired air of rats that received gossypol labeled from 2-¹⁴C-acetate. This indicated that the binaphthalene nucleus of the gossypol molecule was not degraded to CO₂ in the rat. A low level of radioactivity was found in the urine of rats administered either gossypol preparation. In each rat there was radioactivity found in all the tissues that were analyzed; however, the major portion of the radioactivity was excreted in the feces. Paper no. 4189, North Carolina State University Experiment Station, Raleigh, North Carolina. Research described herein represents work done by Charles L. Skutches in partial fulfillment of the requirement for the Ph.D. degree.     

Influence of diet on conversion of14C1-linolenic acid to docosahexaenoic acid in the rat

¹⁴C₁-Linolenic acid was incorporated into lipids of hearts, livers, and carcasses of male rats. We studied the influence of diet composition on extent and distribution of radioactivity. A CHOW diet, a purified, essential fatty acid (EFA)-deficient diet, a purified control diet, and EFA-deficient diets with four fatty acid supplements were used. Supplements of 18∶2n−6, 20∶4n−6, 18∶3n−3, and 22∶6n−3 were given as single doses. Radioactivities in liver phosphatidyl ethanolamines (PE), phosphatidyl cholines, and neutral lipids were measured. The distribution of radioactivity among the fatty acids in liver phospholipids was determined. Rats on CHOW diet incorporated far less radioactivity than any other group into lipids of hearts and livers. Most of the activity in livers was recovered as 20∶5n−3 and 22∶6n−3 in all rats. In EFA-deficient rats, the radioactivity in 22∶6n−3 of liver PE was still increasing 36 hr after¹⁴C₁-linolenic acid had been administered. The n−6 supplements (18∶2n−6 and 20∶4n−6) seemed to reduce the conversion of 20∶4n−3 to 20∶5n−3 (desaturation), whereas the n−3 supplements (18∶3n−3 and 22∶6n−3) reduced the conversion of 20∶5n−3 to 22∶5n−3 (elongation). Formation of 22∶6n−3 may be controlled by 22∶6n−3 itself at the elongation of 20∶5n−3 to 22∶5n−3.     

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.