Ratio of in vivo incorporation of3H arachidonic acid and14C linoleic acid into liver lipids from normal and diabetic rats

Normal and streptozotocin diabetic rats were injected via the portal vein with a labeled solution containing³H arachidonic acid and¹⁴C linoleic acid (³H/¹⁴C ratio, 0.5) 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 percentage distribution of radioactivity into the different lipid fractions was determined. The incorporation of¹⁴C linoleic acid and³H arachidonic acid into liver lipids is apparently reduced in rats with severe diabetes. The higher³H/^14C ratio found in the 1,2 diglycerides from diabetic rats may be explained by the apparently smaller incorporation of¹⁴C linoleic acid or by an isotopic dilution attributable to the great availability of this acid in diabetic rats. On the other hand, the higher³H/¹⁴C ratio observed in triglycerides and phospholipids from diabetic rats, due to a relatively large incorporation of³H arachidonic acid into this fraction, may be explained by the affinity of the enzymes involved in their synthesis for some 1,2-diglyceride units. Insulin was unable to correct the changes observed in the diabetic rats.     

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.     

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.     

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.     

Different metabolism of saturated and unsaturated long chain plasma free fatty acids by intestinal mucosa of rats

During fat absorption, unsaturated long chain fatty acids are esterified at a higher rate than saturated fatty acids of similar chain length. This phenomenon has been attributed to differences in the binding affinity of fatty acids to a cytosolic fatty acid-binding protein. As intestinal mucosa utilizes plasma free fatty acids as well, we investigated whether long chainplasma free fatty acids of different degree of saturation are metabolized also at different rates.³H-Palmitic and¹⁴C-linoleic acid complexed to rat serum were injected rapidly into a tail vein of fasting rats. One, 2 and 4 min later there was no difference between³H and¹⁴C-radioactivity in intestinal mucosa, suggesting equal initial uptake of the two labeled fatty acids from plasma. Despite their equal uptake, the incorporation of the isotopes into ester lipids was significantly different, however: at 2 min, 53.1±3.9% of³H and 73.8±4.6% of¹⁴C were recovered in ester lipids. Phospholipids and triglycerides accounted for most of the mucosal³H and¹⁴C. At 4 min, a similar distribution of isotopes in intestinal mucosal metabolites was found. These data show that despite equal initial uptake by intestinal mucosa unsaturated long chain fatty acids taken up from plasma are esterified to a higher and oxidized to a lower extent than saturated plasma free fatty acids. Unsaturated plasma free fatty acids, therefore, may provide a more important source of fatty acids for endogenous intestinal lipoprotein lipids than saturated plasma free fatty acids. It is speculated that the fatty acid binding protein might be operative not only in the intracellular transport and metabolism of luminal fatty acids but of plasma free fatty acids as well.     

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 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.     

Linoleic and linolenic acid as precursors of the cucumber flavor

Cucumber homogenates were incubated with¹⁴C-linolenic and¹⁴C-linoleic acid. Of the radioactivity, 0.3% was recovered in the fraction of flavor active aldehydes. The distribution of the specific racioactivity indicated that propanal,trans-2-hexenal andtrans-2,cis-6-nonadienal are related to 18∶3 and hexanal andtrans-2-nonenal to 18∶2. A pathway for the development of these compounds is discussed.     

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.     

Lipid metabolism ofAgaricus bisporus (lange) sing.: II. Biosynthesis of sporophore lipids

In vivo biosynthesis of lipids ofAgaicus bisporus, the cultivated mushroom, was studied by examining incorporation of uniformly labeled¹⁴C-linoleic acid and¹⁴C-acetic acid added as supplements to mushroom compost. Incorporation of¹⁴C from both substrates showed similar patterns. Lipids from sporophores were labeled proportionally to their mass with the exception of free sterol which was labeled to a greater proportion in relation to its mass. Quantitative analyses of free sterol was accomplished using pyruvyl chloride 2, 6 dinitrophenyl hydrazone derivatives. Qualitative analysis of free sterol components was made using mass spectrometry. Paper No. 4067 in the Journal Series of the Pennsylvania Agricultural Experiment Station.     

The incorporation of orally fed radioactive γ-linolenic acid and linoleic acid into the liver and brain lipids of suckling rats

The incorporation of radioactivity from orally administered gamma-linolenic acid-1--14C and linoleic acid--3H into the liver, plasma, and brain lipids of suckling rats was studied. Significantly more radioactivity from the former compound was incorporated into the liver and brain lipids 22 hr after dosing. The distribution of the radioactivity in the fatty acids of the liver and brain lipids was different for each isotope. Most of the -3H was still associated with linoeic acid, whereas most of the -14C was in the 20:3 and 20:4omega6 fractions. These results suggest that the desaturation of linoleic to gamma-linolenic acid in vivo is a rate-limiting step in the conversion of linoleic to arachidonic acid.     

Uptake and metabolism of fatty acids by Soybean suspension cells

Soybean suspension cultures very rapidly take up C₁₆ and C₁₈ fatty acids by a nonspecific, nonenzymic binding of exogeneously added fatty acids to cell walls and by a subsequent transfer into the cell where they are rapidly incorporated into triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines.¹⁴C-Palmitic and¹⁴C-stearic acids follow this sequence but are not desaturated, wherease¹⁴C-oleic and¹⁴C-linoleic acids are transferred more rapidly than the saturated fatty acids and are then further modified. All the data fit a sequence of events by which free oleic acid is first activated to a CoA thioester, and then desaturated to linoleyl-CoA; both thioesters are then transferred to triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine.     

Time course of incorporation of 20-carbon polyunsaturated fatty acids in a human keratinocyte cell line

Human keratinocytes (NCTC 2544) in culture were labeled with equal amounts of either¹⁴C-arachidonic acid,¹⁴C-dihomo-γ-linolenic acid or¹⁴C-eicosapentaenoic acid. At various time points, the incubations were stopped and the distribution of the¹⁴C-fatty acids was analyzed. All these eicosanoid precursor fatty acids were effectively incorporated into the cellular lipids of the keratinocytes, and the major radiolabeled individual lipid fraction was phosphatidylethanolamine. The distributions of arachidonic acid and dihomo-γ-linolenic acid within cellular lipids were rather the same. However, less eicosapentaenoic acid than either arachidonic acid or dihomo-γ-linolenic acid was incorporated into the phospholipids and, correspondingly, more eicosapentaenoic acid was incorporated into the nonphosphorus lipids. In the phosphatidylinositol + phosphatidylserine fraction, there was significantly less eicosapentaenoic acid than either arachidonic acid or dihomo-γ-linolenic acid. The present study suggests that these eicosanoid precursor fatty acids are effectively incorporated into the human keratinocytes and that the pattern of incorporation and distribution of eicosapentaenoic acid appears to differ slightly from that of either arachidonic acid or dihomo-γ-linolenic acid.     

Lipid metabolism in plasma,liver, and adipose tissue of rats with experimental chronic nephrotic syndrome

Plasma, liver, and adipose tissue lipid composition and synthesis from [1-¹⁴C] acetate were studied three months following induction of nephrotic syndrome in rats by injection of antiglomerular basement membrane protein. Plasma triglyceride concentrations and specific radioactivities were elevated, and the triglycerides contained increased proportions of oleic acid. Plasma cholesterol and phospholipid concentrations were also increased, but free fatty acid levels were not. Liver triglyceride concentrations were decreased and incorporation of [1-¹⁴] acetate into liver triglycerides was also depressed below that of normal controls. Nephrotic rat liver triglycerides contained a higher proportion of oleic acid and lower arachidonic acid than did controls. Incorporation of [1-¹⁴C] acetate into adipose tissue lipids of the nephrotic rats was increased, and the proportion of palmitic acid was decreased. In the chronic nephrotic rat, the major source of the increased plasma triglycerides may be fatty acids mobilized from adipose tissue stores.     

Origin of the high saturated fatty acid content of rat fecal lipids

The biohydrogenation of unsaturated fatty acids and the preferential absorption of unsaturated fatty acids over long chain saturated fatty acids from the gut have been investigated to find the origin of the high saturated fatty acid content of the facal lipids of rats fed soybean oil. Label from dietary (1-¹⁴C)-linoleic acid was recovered in the saturated and monounsaturated fatty acids of the fecal lipid. However, when (9,10-³H)-stearic acid and (1-¹⁴C)-linoleic acid were fed together, the isotope ratio (³H/¹⁴C) of the fecal lipid was 1.9 times that of the diet. It is concluded that both processes occur.     

Metabolism of14C-labelled oleic acid,erucic acid and nervonic acid in rats

1-¹⁴C-Oleic acid, 2-¹⁴C-erucic acid and 2-¹⁴C-nervonic acid were administered to rats by tail-vein and the distribution of radioactivity in liver lipids was determined at intervals from 15 min to 6 hr after injection. High levels of activity were found after short time intervals which were mainly associated with triglycerides in the case of oleic acid and with free fatty acids in the case of erucic acid and nervonic acid. The activity in these lipids decreased with time and was later exceeded by that in more polar lipids. In rats given erucic acid or nervonic acid, sphingolipids were more highly labelled than glycerophosphatides. Nervonic acid showed little tendency to form a complex with serum albumin and erucic acid complexed less readily than palmitic acid. Presented at the AOCS Meeting in Houston, April 1965.     

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.     

Zinc deficiency increases the rate of Δ6 desaturation of linoleic acid in rat mammary tissue

The effect of zinc deficiency on the Δ⁶-desaturation of [1-¹⁴C] linoleic acid was studied in mammary tissue microsomes from lactating rats. The rats were maintained on zinc-adequate (20 ppm zinc) or zinc-deficient (10 ppm zinc changing to 0.5 ppm zinc during last trimester) diets throughout gestation and for the first 3 days of lactation. Mammary tissue microsomes were incubated with [1-¹⁴C] linoleic acid and other samples of mammary tissue, mammary milk and the milk in the stomachs of the pups were analyzed for total fatty acid composition. In mammary microsomes from zinc-deficient rats, Δ⁶-desaturation of linoleic acid was 3.4 times greater than in microsomes from zinc-adequate rats. This change in metabolism of linoleic acid was reflected by comparable changes in the relative tissue and milk composition of linoleic and arachidonic acids and in the ratios of palmitic to palmitoleic acid, stearic to oleic acid and linoleic and arachidonic acid.     

Effects of sodium butyrate on the transfer of arachidonic acid to phosphatidylcholine in a clonal oligodendrocyte cell line (CB-II)

The effect of sodium butyrate on membrane phospholipid metabolism in a neonate rat cerebellum derived clonal oligodendrocyte cell line (CB-II) was investigated. Sodium butyrate is an agent known to induce cell differentiation and morphological transformations. A comparison of thein vivo phospholipid labeling patterns obtained by incubating CB-II cells with [³H]choline, [¹⁴C]myristic acid or [³H]arachidonic acid indicated that butyrate altered the route of acylation-deacylation in phosphatidylcholine (PC) biosynthesis. Using anin vitro incubation system containing homogenates of CB-II cells, the largest proportion of radioactivity was found in PC, and addition of sodium butyrate resulted in a further increase in the transfer of arachidonic acid to PC, but not to phosphatidylinositol. Similar results were obtained when thisin vitro acylation activity was tested using homogenates from sodium butyrate pretreated cells. The butyrate effect was observed regardless of whether or not exogenous lysophosphatidylcholine (LPC) was added to the incubation system. Addition of butyrate did not result in a change in the activity of LPC:acyl-CoA (coenzyme A) acyltransferase (EC 2.3.1.23) in CB-II cells upon incubating cell homogenates with [1-¹⁴C]arachidonoyl-CoA and LPC. However, when cell homogenates were incubated with [³H]arachidonic acid in the presence of 2.5–10 mM sodium butyrate, arachidonoyl-CoA synthesis was stimulated. A time course study demonstrated that significant stimulation occurred after three minutes. Taken together, the results suggest that in CB-II cells, sodium butyrate stimulates the transfer of arachidonic acid into PC and that this effect is at least partially due to a stimulation of arachidonoyl-CoA ligase (EC 6.2.1.3).