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.     

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.     

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.     

Lipid binding in mitochondria from testes of normal and essential fatty acid deficient rats

Essential fatty acid (EFA) deficiency in rat causes severe degeneration of spermatogenic tissue. Previously it was shown that the distribution of lipid classes changes very little during tissue degeneration. However it is well known that the fatty acid spectrum in lipids from testicular tissue is altered drastically during EFA deficiency. The molecular binding of lipids in membrane structures might be altered when a larger amount of ω9-acids is present in the various lipid classes in testes of EFA-deficient rats. In the present studies comparison was made of the binding of lipids in testicular mitochondrial membranes from rats fed a fat-free diet or a diet containing 6% peanut oil for 26 weeks. Isolated mitochondria were coated on glass beads, then dried and packed into a column, whereafter the membrane lipids were eluted with solvents with increasing dielectric constants. The differences between the binding of lipid classes in supplemented and EFA-deficient rats were not pronounced, but a tendency to a weaker binding in the EFA-deficient rats was observed. However for both groups the various extracts showed marked differences in the distribution of lipid classes concurrent with the change of the eluent. This indicates a different kind of binding in the membrane, not only for different lipid classes, but also within a special lipid class. Thus both phosphatidylcholines (PC) and phosphatidylethanolamines (PE) were found in extracts with quite different dielectric constants. The fatty acid composition of PC and PE in the major fractions eluted with chloroform and ethanol, respectively, was essentially the same. This indicates that the successive release of phospholipids (PL) in these two fractions was not based on fatty acid solubility properties but on variable binding in the membrane structure. The introduction of ω9-polyenoic fatty acids instead of ω6-polyenoic fatty acids in the PL of mitochondria membranes from EFA-deficient rats seems to be the only deviation in the lipid pattern of EFA-supplemented and EFA-deficient animals, and might therefore be responsible for the symptons of EFA deficiency. Presented in part at the AOCS-ISF World Congress, Chicago, September, 1970.     

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.     

Composition and concentration of lipoproteins in the serum of normal rats and rats deficient in essential fatty acids

A comparison is made of the concentration and chemical composition of serum lipoproteins of normal rats and rats deficient in essential fatty acids. The concentration of very low density lipoproteins (VLDL) and of low density lipoproteins (LDL) in serum of deficient rats is about half that found in normal rats, but the concentration of high density lipoproteins (HDL) is higher than normal and they contain an increased amount of cholesterol esters. The proportion of cholesterol that is esterified is much greater than normal in the serum of deficient rats. The deficiency of essential fatty acids also appears to result in compensating changes occurring in the composition of serum lipoproteins. In both VLDL and LDL of deficient rats the proportion of protein is raised and that of phospholipid lowered compared to normal, while the proportions of trigly ceride and cholesterol esters are unchanged.     

In vitro incorporation of 1-14C nonanal-9-oic acid into plasma and human red blood cells lipids

Nonanal-9-oic acid is incorporated principally into plasma phospholipids, whereas oleic acid is incorporated into red cells. This incorporation does not require the presence of adenosine 5′-triphosphate and Coenzyme A and is carried out in the absence of red cells. the incorporation of nonanal-9-oic acid in blood lipids takes place in the first 10 min of incubation.     

In vivo incorporation of labeled fatty acids in rat liver lipids after oral administration

Striking differences were found in the compartmentalization of fatty acids into liver lipid fractions. The saturated fatty acids—lauric, myristic, palmitic and stearic—were incorporated into phosphoglycerides at faster rates with increasing chain lengths, while triglyceride incorporation was almost uniform. The degree of incorporation of the unsaturated fatty acids into phosphoglycerides (structural) compared to triglyceride (storage and energy) was the converse of their oxidation rates. The incorporation of oleic, linoleic and α-linolenic acids was mainly into triglyceride, whereas dihomo-γ-linolenic acid and arachidonic acid were preferentially incorporated into phosphoglycerides. The data suggest that distribution of each fatty acid is different depending on its destination for structural or energy function.     

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.     

The incorporation of fatty acids of different chain length into liver and biliary lipids in the perfused rat liver

In an attempt to correlate the incorporation of fatty acids (FA) of different chain length into liver and biliary lipids’ isolated rat livers were perfused for 2 h with Krebs-Ringer bicarbonate containing 1% albumin and 10 μmol of [1-¹⁴C]-labeled FA: C₂’ C₈’ C₁₀’ C₁₂’ C₁₆’ and C_18∶1. One to 1.36 μmol of medium-chain fatty acids (MCFA’ C₈’ C₁₀’ and C₁₂) and 6.6 μmol of long-chain FA (LCFA) were incorporated into liver lipids’ 40% of the latter into phosphatidylcholine (PC). ¹⁴C-acetate (13 nmol) was incorporated into biliary cholesterol; ¹⁴C-MCFA contributed only 3.2–5 nmol; LCFA did not lead to newly synthesized cholesterol. Newly synthesized liver PC (2.75 to 3.25%) and newly synthesized liver cholesterol (6.5 to 10%) were secreted into bile. The specific radioactivity of biliary PC after infusion of all-saturated FA was 3.8–6.8 times higher than that of liver PC; for C_18∶1 it was only 1.7-fold. The specific radioactivity of biliary cholesterol’ as compared to liver cholesterol’ was 12 times higher for C₂ and five times higher for MCFA. This indicates that a considerable proportion of the newly synthesized lipids was secreted into bile prior to significant mixing with preexisting liver PC and cholesterol pools. liver PC contained 8% of unchanged ¹⁴C−C₁₂; while ¹⁴C−C₁₀ was not detected. Biliary PC’ in contrast’ contained 18% of unchanged ¹⁴C−C₁₂ and 3% ¹⁴C−C₁₀. These results suggest that after prolonged infusion of medium-chain triacylglycerols/longchain triacylglycerols to patients’ biliary PC may become enriched with MCFA. In addition’ the oxidation of these FA may provide C-2 units which increase cholesterol synthesis.     

Essential fatty acid supplemented diet increases renal excretion of prostaglandin E2 and water in essential fatty acid deficient rats

Weanling male rats were fed an essential fatty acid (EFA)-deficient diet for 25 weeks and then switched to an EFA-supplemented diet for 3 weeks. Control rats received the EFA-supplemented diet for 25 weeks and then the EFA-deficient diet for 3 weeks. Throughout the last 19 weeks, the rats were housed in metabolic cages once a week for a 24-hr period. Urinary excretion of prostaglandin E₂ (PGE₂) was estimated by radioimmunoassay. Throughout a period of 12 weeks (weeks 13–24) water consumption increased ca. 60%, and urine output and PGE₂ excretion decreased ca. 45% and 70%, respectively, in the EFA-deficient rats. Feeding EFA-supplemented diet to the EFA-deficient rats for 3 weeks decreased the water consumption and raised the urine output to that observed in the controls. However, the urine output was corrected within 1 day whereas the water consumption was not corrected until the second measurement 8 days after the dietary change. The PGE₂ excretion increased more than 9-fold (from 18±8 ng/24 hr to 165±51 ng/24 hr) 1 day after EFA-supplementation, followed by a decrease to 86±29 ng/24 hr over the following 2 weeks. On the basis of the present data, it is suggested that EFA deficiency in rats causes diminished PGE₂ excretion, which can be normalized by EFA supplementation. The normalization of the urine flow may, in part, be caused by the concomitant considerable increase in endogenous PGE₂ synthesis. Presented in part at the International Symposium on Prostaglandins and the Kidney, Stuttgart, West Germany, July 1980.     

Fatty acid composition of liver lipids in rats fed brominated fatty acids

Feeding rats diets containing brominated corn oil or di- or tetrabromostearate as the monoglyceride produced changes in fatty acid composition of liver lipids. Those changes associated with the feeding of brominated corn oil or tetrabromosterates could be explained by the accumulation of triglyceride, and the changes associated with the feeding of dibromostearate could result from the proliferation of a membrane system. A unique response to the feeding of diets containing brominated corn oil is an increase in the level of γ-linolenic acid.     

The effect of the environmental temperature on the fatty acid composition and on thein vivo incorporation of 1-14C-acetate in goldfish (Carassius auratus L.)

Two-year-old goldfish were adapted to 10C and 35C environmental temperatures during a three-week period, and the fatty acids from triglycerides and certain phospholipids were analyzed by gas-liquid chromatography. Over-all unsaturation of the major fatty acids increased with lower temperature in all lipids which were examined although fish maintained at 10C actually had less polyenoic acid in their tissues than did those maintained at 35C. Fish acclimated to 10C and 30C were injected with 1-¹⁴C-acetate, and the activities of the isolated fatty acids were counted. The incorporation of¹⁴C into the fatty acids was much greater at the lower temperature. A comparison of the activities of saturated and unsaturated fatty acids within each temperature group revealed a tendency toward higher incorporation into the unsaturated acids at lower temperature. The possible correlations between accelerated biosynthesis of polyenoic acids and the lower tissue levels of these acids in the cold-adapted fish are discussed. These studies were supported in part by Contract AT(04-1) GEN-12 between the Atomic Energy Commission and the University of California. Supported in part by PHS Research Career Award No. GM-K6-19, 177, from the Division of General Sciences, National Institutes of Health.     

Increased liver lipase activity in rats with essential fatty acid deficiency

Liver lipase activity was measured in EFA-deficient rats (long-term) and in control rats and rats fed an EFA-deficient diet for two weeks (short-term). Liver lipase activity was significantly enhanced by EFA deficiency, both in long-term and short-term experiments. The enhanced liver lipase activity could be normalized by feeding these rats normal laboratory chow for 14 days. Since during EFA deficiency prostaglandin synthesis is impaired, the possible involvement of prostaglandins in the observed changes in liver lipase actvity during EFA deficiency was studied. Administration of the prostaglandin synthesis inhibitor indomethacin (5 mg/kg body weight, i.p.) to normally fed rats for two days led to an increase of liver lipase activity. Prostaglandin E₂ was found to inhibit the secretion of liver lipase activity by freshly isolated parenchymal liver cellsin vitro. These results indicate that the increase in liver lipase activity during EFA deficiency may be due to an impairment of the prostaglandin synthesis.     

Metabolism of 1-14C linolenic acid in developing brain: II. Incorporation of radioactivity from 1-14C linolenate into brain lipids

Metabolism of 1-¹⁴C linolenic acid was studied in growing animals by injecting the tracer intraperitoneally into 12–13 day old suckling rats and following up the results by sacrificing groups of animals at 8 hr, 48 hr, 15 day, and 45 day intervals. In the first 15 days, there was a greater decrease in radioactivity of brain total lipids compared to the later period, although the earlier age period is characterized by lipid deposition rather than breakdown. Since the 18∶3 ω3 family of fatty acids occurs largely in the brain total phosphatidyl ethanolamine fraction, we expected that, in the initial period, total phosphatidyl ethanolamine would be the most highly radioactive component. However, results showed that 8 hr after the tracer phosphatidyl choline had the highest specific radioactivity. When the total phosphatidyl ethanolamine fraction was resolved into diacyl and alk-1-enyl species, it was found that radioactivity was not distributed evenly between the two species. There was a progressive increase in radioactivity of the alkenyl and a decrease in the diacyl species. Forty-eight hr after the tracer, however, the radioactivity of phosphatidyl ethanolamine increased and at 45 days remained slightly higher than phosphatidyl choline. Radioactivity of cholesterol, a result of synthesis from acetate undoubtedly derived from the breakdown of tracer linolenate, was also high 48 hr after tracer and remained high until 45 days.     

Rat testicular lipids and dietary isomeric fatty acids in essential fatty acid deficiency

Weanling rats were fed essential fatty acid-deficient diets, either completely fat-free, or with partially hydrogenated fish oil (PHFO, 28 wt %), or with fractions derived from PHFO containing primarily positional isomers oftrans-eicosenoate (20∶1, 3 wt %) ortrans-docosenoate (22∶1, 3 wt %). Control animals were fed a peanut oil-containing diet (28 wt %). After 5 or 15 weeks on the diet, the content of neutral and phosphorus-containing lipids in the testes was determined. The fatty acid distribution in major lipid classes was analyzed for animals fed the diets for 15 weeks. The testicular stage of maturation or degeneration was assessed by histology. The group fed PHFO exhibited signs of complete testicular degeneration, or lack of maturation, already after 5 weeks, whereas the animals on the diets with the very long chain monoenoic acids suffered severe degenerations only after 15 weeks. In the PHFO-fed rats, a sharp decline in the concentration of testicular triacylglycerols was observed. In all of the essential fatty acid-deficient groups, an increase in testicular sphingomyelin was observed. Cholesterol levels were fairly similar among all dietary groups. The total testicular fatty acids of the PHFO-fed animals contained somewhat more eicosadienoic acid than found in the other groups, and somewhat less (n−9)-acids. In all EFA-deficient groups, (n−6)-acids were lowered, in particular in triacylglycerols and phosphatidyl cholines. The PHFO group did not show a lower (n−6)-concentration than the other deficient groups, in spite of the more severe symptoms of deficiency. There was no evidence of a major accumulation of long chain isomeric fatty acids in the degenerated testes of the PHFO-, 20∶1, and 22∶1-fed groups.     

Retention of linoleic acid in carcass lipids of rats fed different levels of essential fatty acids

Rats of an inbred Sprague-Dawley strain were fed purified diets with low (0.3% of total energy), normal (3%) or high (10%) content of essential fatty acids (EFA) for at least three generations. Two 30-days-old rats with similar weights were chosen from one litter. One was killed; weight increase and food consumption of the other rat was measured for 15 days. Total lipid content and fatty acid composition in total lipid and lipid classes were determined in both rats. Seven pairs of rats from each group were treated in the same way. Calculations based an amount of linoleic acid ingested and retained in the carcass lipids showed that 50% of the ingested linoleic acid was retained in the low EFA rats compared to 10–15% in the normal and high EFA rats.     

In vitro biosynthesis of prostaglandin E2 by kidney medulla of essential fatty acid deficient rats

Weanling rats were fed either a semi-synthetic diet with no fat, with 28% by wt partially hydrogenated fish oil, or with 28% by wt arachis oil (control diet) for 6 or 71/2 months. The in vitro conversion of arachidonic acid to prostaglandin E₂ by homogenates of the rat kidney medulla was measured by gaschromatography with electron capture detection. The kidney medulla of essential fatty acid deficient animals showed increased activity for the in vitro conversion of exogenous arachidonic acid to prostaglandin E₂ when compared to the controls. The change of the enzymatic activity in the essential fatty acid deficient animals was reversible, as shown by refeeding. Inhibition of the prostaglandin synthetase was found at exogenous substrate concentrations higher than 50–100 μM.