Measurement of the incorporation of orally administered arachidonic acid into tissue lipids

The applicability of a stable isotope method to monitor the mixing of dietary arachidonic acid with endogenous arachidonic acid in tissue lipids was evaluated. Rats were fed octadeuterated arachidonic acid during a 20-day period, and the entry of the dietary acid into lipid esters of various tissues was examined by gas chromatographymass spectrometric (GC-MS) analysis of their fatty acids. The rats were maintained on a fat-free diet from weaning until 63 days old to enhance the ratio of the dietary acid to endogenous arachidonate. Three separate forms of eicosatetraenoic acid in the tissue lipids could be distinguished by GC-MS: octadeuterated arachidonic acid (recent dietary origin), unlabeled arachidonic acid (maternal origin) and unlabeled, 4,7,10,13-eicosatetraenoic acid (originating from palmitoleic acid). The total eicosatetraenoic acid in the tissue lipids contained about 90% arachidonate from recent dietary origin in lung, kidney, heart and fat, 70% in muscle and liver and 27% in brain. The n−7 isomer of eicosatetraenoic acid was estimated to make up 6% or less of the total eicosatetraenoic acid in lung, kidney, brain, muscle and heart tissue lipids, but it comprised around 15% of the total eicosatetraenoic acid in liver. The unlabeled arachidonic acid of maternal origin thus comprised only about 10% of the eicosatetraenoic acid in all tissues examined except muscle and brain, where it was 24% and 70% of the eicosatetraenoic acid, respectively. The relative amounts of the three forms of eicosatetraenoic acid are consistent with a limited access of dietary arachidonate to the brain tissue and with a competition between the dietary n−6 isomer and the endogenous n−7 isomer for esterification in the liver. Because most muscle mass would have formed after weaning, the high proportion of maternal arachidonate in the muscle lipids suggested that maternal arachidonate may have been displaced from other tissues to muscle, from which it equilibrated slowly with dietary arachidonate acid. The combination of deuterated arachidonic acid and GC-MS analysis thus furnished more detailed information about the composition and origin of eicosatetraenoic, acid in tissue lipid esters than that previously available from radiotracer studies or GC-MS analyses alone.     

Incorporation of conjugated linoleic acid and vaccenic acid into lipids from rat tissues and plasma

The objective of this study was to determine the incorporation of conjugated linoleic acid (CLA) into triacylglycerols (TAG) and phospholipids (PL) of tissues and plasma, and to interpret the role of dietary‐derived vaccenic acid (VA) in increasing the tissue content of CLA (c9,t11) and the influence on the fatty acid profile. We fed five groups of rats semi‐purified diets with varying levels of CLA and VA: control butter with low CLA (c9,t11) and VA; control butter added 5% CLA (c9,t11); control butter added 5% Tonalin [equal amount of CLA (c9,t11) and CLA (t10,c12)]; control butter added 5% VA; butter with high CLA (c9,t11) and VA (H‐CLA), for 3 weeks. The highest incorporation of CLA (c9,t11) was found in adipose tissue, and the lowest was observed in liver. Low intake of CLA (c9,t11) combined with high intake of VA resulted in a higher incorporation of CLA (c9,t11) in tissues due to the conversion of VA to CLA (c9,t11), compared to feeding CLA (c9,t11) without VA. However, in enterocytes, the proportion of CLA (c9,t11) was low after feeding VA, indicating no or only a minor conversion of VA to CLA (c9,t11) in the intestine. The incorporation of CLA (t10,c12) into TAG from plasma and tissues was generally much lower than that of the CLA (c9,t11) isomer, except in the enterocyte TAG, which had similar proportions of the two isomers.     

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.     

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.     

Assimilation of dietary eicosenoic and erucic acid esters

1. Eicosenoic acid, fed to rats as methyl ester to the extent of 5% by weight of the diet, was deposited in substantial amount in the body fat.
2. Erucic acid, fed in the same way, was deposited in considerably lesser amount.
3. Neither of these acids appeared to be as readily deposited in body fat as the linoleic acid of corn oil.
4. The three monounsaturated acids, oleic, eicosenoic, and erucic, were apparently deposited without alteration in chain length or degree of saturation. There was no evidence of conversion of eicosenoate to arachidic or stearic acids, or of erucate to behenic, arachidic, or stearic acids in any appreciable amount. Similarly there appeared to be little or no conversion of eicosenoate to oleic acid or of erucate to eicosenoic acid.
5. The eicosenoate in the diet was not converted to arachidonic acid by the animal.
6. The amount of total fat deposited and the proportion in carcass, skin, and viscera were approximately the same on diets of oleate, eicosenoate, and erucate.

     

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.     

Docosahexaenoic acid: Nutrient and precursor of bioactive lipids

Docosahexaenoic acid (DHA), the end‐product of the n‐3 family fatty acid, is an abundant component in the brain phospholipids, and a major nutrient of marine lipids. It is accumulated in this compartment from the non‐esterified pool bound to blood plasma albumin. 1‐Lyso,2‐DHA‐glycerophosphocholine is another form, also bound to albumin, which appears even more efficient than non‐esterified DHA for its brain accretion. DHA, as a highly unsaturated fatty acid, may affect the cell redox status. As expected, it may promote lipid peroxidation at high concentrations, but most interestingly may prevent it at low concentrations. DHA is also a fairly good substrate of lipoxygenases, especially the n‐9 and n‐6 ones. Hydroxy derivatives, i.e., docosanoids, exhibit potent biological activities, which may explain part of the potential benefit of DHA in the brain and vascular bed.     

Monitoring the oxidation of docosahexaenoic acid in lipids

The oxidation of free DHA, DHA mixed with PC, and DHA incorporated into PC, PE, or TG was evaluated to determine which lipid provided DHA with the best protection against oxidation. DHA was either situated at the sn-1 position, sn-2 position, or both positions of the phospholipid, whereas the TG contained DHA at all positions. All lipids were incubated as bulk lipids, in chloroform, or as an emulsion in contact with air at 25–30°C for 28 d. Since DHA, which is highly sensitive to oxidation, has a great impact on our health and is desired as a food additive, the stability of this FA is of great importance. This study was mainly focused on the primary oxidation products, which were monitored as eight monohydroperoxy-DHA isomer groups, the total amount of polyhydroperoxides, and the PV. However, a measure of secondary oxidation products, the carbonyl value, was also monitored. We found that DHA was most protected against hydroperoxide formation when it was incorporated at one position of either PC or PE. In these lipids, hydroperoxide formation at carbon atoms 4, 7, 8, and 11 was completely prevented. DHA mixed with PC was also protected, although to a lesser extent, and all hydroperoxide isomers were detected. In contrast, PC and TG containing DHA at all positions should be avoided, since they were highly oxidized.     

Preservation of lipids with malic acid

This paper deals with an evaluation of malic acid in a number of different lipids of animal and vegetable origin under comparable conditions. An Active Oxygen Method was used, with citric acid as the standard of comparison. In most cases, the performance of the acids was determined in the presence and absence of added iron salts and antioxidants butylated hydroxyanisole and butylated hydroxytoluene. Mathematical evaluation of the data shows malic acid to be at least as effective as citric acid in most cases. In very few instances, a reversal of performance was observed depending on experimental conditions used. In a few experiments, malic acid was also tested against phosphoric acid and found to be equal to or better than the latter. Phosphoric acid caused darkening of the oils at the test temperature used.     

Tumor lipids: Long chain dienoic acid in sphingomyelin

The fatty acids derived from sphingomyelin fractions of 10 rat and mouse tumors were examined quantitatively. All tumors, except a nontransplantable fibroadenoma, contained significant levels of a C-24 dienoic acid which is absent from sphingomyelin of normal tissues or present in only trace amounts. The data suggest an abnormality in the metabolism of sphingolipids of tumor cells.     

Fatty acid distribution in lipids and32P incorporation into phospholipids during early amphibian development

Several aspects of lipid composition and³²P incorporation were studied during early embryogenesis of the toad,Bufo arenarum, Hensel. The surveyed stages ranged from unfertilized oocyte to neural tube formation. The fatty acid distribution in polar and neutral lipids, as well as in acetone eluate from Unisil columns was similar in unfertilized oocyte and late blastula stage. There was no significant effect of cell cleavage on the fatty acid composition of these lipid fractions. Neutral lipids represent ca. 67% of the total lipids. The main components of the phospholipids were phosphatides of choline and ethanolamine. The total lipid and phospholipid content does not change through the studied stage of neurula. However a large increment in the phospholipid's specific radioactivity occurs when³²P is injected along with the hormone to induce ovulation. It is suggested that this may reflect changes in turnover rates rather than net biosynthesis. Since a large amount of cell membranes is being formed during the early development and because the level of phospholipids remains constant, an explanation is offered regarding membranogenesis. Active phospholipid biosynthesis may take place during oogenesis. These lipids may be stored in the yolk platelet, and fertilization may regulate the functioning of a transport mechanism to corresponding membrane sites. The increased incorporation of³²P may reflect changes in the activity of new membranes.     

Interference of polar lipids with the alkalimetric determination of free fatty acid in fish lipids

An examination of the suitability of an alkalimetric method for the determination of free fatty acid (FFA) contents in fats, oils, and lipid extracts was conducted by comparing AOCS method Ca 5a-40 with a method based on a Chromarod-latroscan thin-layer chromatography-flame-ionization detector (TLC-FID) system. The FFA contents determined by the alkalimetric method were consistently higher than the genuine FFA contents obtained by the latroscan TLC-FID method. Phospholipids were found to be the major components that contributed to the alkali-titratable, nongenuine FFA in the total FFA determined alkalimetrically. Contributions from other polar lipid components were smaller, but they dominated as the proportion of phospholipids fell. The other alkali-titratable polar components may include oxidized lipids and their by-products bound to protein fragments. The accurate determination of FFA contents by alkalimetric methods may only be applicable to those commercially refined fats and oils that contain negligible amounts of phospholipids. Corrections for the alkalimetrically determined FFA contents should be made for those fats and oils with relatively high phospholipid contents by correlating the nongenuine FFA contents and the phospholipid contents.     

Incorporation of acetate into fatty acids and complex lipids of soybean cotyledon slices under aerobic and anaerobic conditions

Soybean slices incubated with [1-¹⁴C] acetate in the presence of air synthesized fatty acids to a greater extent than did slices in the absence of air. The proportion of radioactive fatty acids incorporated into the neutral lipid was ca. 35% in the presence or absence of air. However, both the proportion and the absolute amount of radioactive fatty acids in phospholipids were greater in the presence of air. This difference was particularly great in phosphatidic acid and in a minor uncharacterized phospholipid component. Significant incorporation of acetate into monoenoic acids was observed in these two lipids and in phosphatidyl choline. The latter also showed an accumulation of newly synthesized polyenoic acids when air was present. Stearic acid synthesis was greater under aerobic than under anaerobic conditions. The present results support the concept that a relationship exists between the synthesis of unsaturated fatty acids and their incorporation into phospholipids in plants.     

Studies on the fatty acid composition of crayfish lipids

The fatty acid composition of carcass and exoskeleton lipids was determined for the freshwater crayfishOrconectes rusticus. Lipid fractions were isolated by column and thin-layer chromatography. Fatty acid methyl esters and alcohol acetates were then prepared and analyzed by gas-liquid chromatography. Peak identities were established from retention time data for methyl esters, hydrogenated methyl esters, and saturated, monoene, diene, and polyene methyl esters separated as acetoxy-mercuri-methoxy derivatives. Minor component acids were estimated from their relative compositions in these fractions. Presented at the symposium honoring J. B. Brown, AOCS meeting in Chicago, 1964.     

Fatty acid metabolism in L1210 murine leukemia cells: Differences in modification of fatty acids incorporated into various lipids

L1210 leukemia cells can utilize all of the main fatty acids that normally are present in the ascites fluid in which they grow. This finding is consistent with the view that L1210 cells derive most of their fatty acids from the ascites fluid. From 80–90% of each fatty acid was incorporated into cell lipids without structural modification, suggesting that the lipid composition of these cells can be altered by changing the type of fatty acids to which they are exposed. Most importantly, the palmitate that was subsequently incorporated into total cell phospholipids was elongated and desaturated somewhat more than that incorporated into triglycerides. This difference was due primarily to more extensive modification of the palmitate incorporated into the ethanolamine phosphoglycerides fraction. Although there was no difference between total phospholipids and triglycerides with linoleate, more of the linoleate incorporated into ethanolamine phosphoglycerides was elongated and further desaturated than that incorporated into choline phosphoglycerides and triglycerides. These findings indicate that fatty acids incorporated into various cell lipid fractions are not structurally modified to the same extent. There appears to be greater modification of fatty acid used for ethanolamine phosphoglyceride synthesis as compared with triglyceride and choline phosphoglyceride synthesis.     

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.     

cis-Vaccenic acid in mango pulp lipids

A peak corresponding to a methyl octadecenoate other than oleate has been detected on the capillary gas chromatogram of the methyl esters of mango pulp fatty acids. This octadecenoate was isolated by silica gel and argentation column chromatography, high performance liquid chromatography and argentation thin layer chromatography, and then analyzed by infrared, nuclear magnetic resonance and gas chromatography-mass spectrometry, chromatographic separations and oxidative degradation. These analytical data proved that the octadecenoic acid wascis-vaccenic acid (cis-11-octadecenoic acid). The concentration of this acid in total octadecenoic acids ranged from 35% to 50% in the pulp of mangoes from Fiji, Mexico, the Philippines and Taiwan.cis-Vaccenic acid was revealed to be one of the major component fatty acids of non-polar lipids (mainly triacylglycerols), glycolipids and phospholipids in mango pulp. The glycolipids containedcis-vaccenic acid (ca. 20%) in higher concentration than oleic acid (ca. 15%). A trace amount ofcis-vaccenic acid (0.5%) was detected in the total lipids of mango seeds. Profile of fatty acid composition of mango pulp lipids (0.2–0.3 wt% of wet pulp) was characterized by the presence of n−7 acid isomers,cis-vaccenic acid and palmitoleic acid, and unusual mono- and dienoic positional isomers.     

Intake of different eicosapentaenoic acid-containing lipids and fatty acid pattern of plasma lipids in the rats

The ethyl ester of eicosapentaenoic acid (EPA) is the only pure EPA-containing lipid available in bulk for oral administration. However, there is doubt as to whether EPA ethyl ester can efficiently increase the plasma levels of EPA in comparison with the ability of other kinds of EPA-containing lipids to do so. Therefore, two other kinds of EPA-containing lipids were prepared to study the efficiency of oral administration of those lipids for increasing the EPA content in plasma phospholipids and cholesteryl esters. EPA-containing lipids which were investigated were [A], 1,2,3-trieicosapentaenoyl-glycerol, [B] 2-eicosapentaenoyl-phosphatidylcholine and [C] ethyl ester of EPA. An adjusted amount of lipids [A], [B] and [C] was administered to rats through a gastric tube for 4 days (the first experiment) or for 10 days (the second experiment), and the fatty acid composition of plasma phospholipids and cholesteryl esters was determined. In the first experiment, there were no significant differences in the efficiency for increasing EPA levels in either phospholipids or cholesteryl esters among the lipids. In the second experiment, the EPA levels of both plasma phospholipids and cholesteryl esters of rats administered ethyl ester of EPA were significantly higher than those of rats administered 2-eicosapentaenoyl-phosphatidylcholine. The EPA levels of the rats administered 1,2,3-trieicosapentaenoylglycerol were between the levels of the two groups mentioned above, but the differences in the EPA levels were not significant. Although an ethyl ester-type molecule is not a naturally occurring lipid, ethyl ester of EPA is equal to 1,2,3-trieicosapentaenoyl-glycerol and appears to be superior to 2-eicosapentaenoyl-phosphatidylcholine as to the efficiency for increasing EPA levels in total plasma phospholipids and plasma cholesteryl esters.     

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.