Metabolism of linolenic acid in developing brain: I. Incorporation of radioactivity from 1-14C linolenic acid into brain fatty acids

Twelve-thirteen day old rats were given 1-¹⁴C linolenic acid by intraperitoneal injection. Fatty acids were isolated from the brains of animals sacrificed at the end of 8 and 48 hr and 15 and 45 days. Eight hr after the tracer, radioactivity was found neither in 18∶3 nor its endproduct, 22∶6, and palmitate was the most highly radioactive component. At longer intervals, 22∶6 seemed to retain much of the radioactivity, whereas palmitate showed a precipitous decline in radioactivity. Initial oxidation of linolenate and sparing of the linolenate complexed with polar lipids are discussed.     

Biosynthesis of polyunsaturated fatty acids in the developing brain: I. Metabolic transformations of intracranially administered 1-14C linolenic acid

Thirteen-day old rats were given intracranial injections of 1-¹⁴C linolenic acid (allcis 9,12,15 octa decatrienoic acid) and were sacrificed after 8 hr. Analysis of brain fatty acids showed that 16∶0, 18∶0, 18∶1, 18∶3, 20∶3, 20∶4, 20∶5, 22∶5, and 22∶6 were labeled. The total fatty acid methyl esters were separated into classes according to degree of unsaturation on a AgNO₃∶SiO₂ impregnated plate. The bands were scraped off and the eluted fatty acids were first analyzed by radiogas liquid chromatography and then subjected to reductive ozonolysis to determine double bond position. The saturated acids, 16∶0, and 18∶0, as well as the mono-unsaturated 18∶1, must have been formed from radioactive acetate produced by β oxidation of the injected linolenate. Among the polyunsaturated fatty acids, the triene fraction was characterized and identified as 18∶3 ε3 (Δ^9,12,15), the starting material, and 20∶3 ω3 (Δ^11,14,17); the tetraene fraction was identified as 20∶4 ω3 (Δ^8,11,14,17); the pentaene fraction was identified as 20∶5 ω3 (Δ^5,8,11,14,17) and 22∶5 ω3 (Δ^{7,10,13,16,19}); and, finally, the hexaene fraction was shown to be 22∶6 ω3 (Δ^{4,7,10,13,16,19}). The biosynthesis of these ω3 family fatty acids in the brain in situ is discussed.     

Incorporation of [1-14C]octadecanol into the lipids ofLeishmania donovani

After incubation of stationary phaseLeishmania donovani with [1-¹⁴C] octadecanol, about 70% of the precursor was taken up within 3 hr. Wax esters and acyl moieties of glycerolipids contained most of the¹⁴C-activity from 3 to 6 hr, because octadecanol was partly oxidized to stearate. Ether moieties were only weakly labeled. After 40 hr, 1-0-aklyl and 1-0-alk-1′-enyl diacylglycerols as well as 1-0-alkyl and 1-0-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines contained nearly all of the radioactivity. Most of the label in the neutral ether lipids was located in the alkyl ether side chain, whereas, in the phosphatidylethanolamine fraction, most of the label was found in the alkenyl ether side chain. Administration of 1-0-[1-¹⁴C] hexadecyl glycerol resulted in rapid labeling of the vinyl ether side chain of phosphatidylethanolamine plasmalogen (1 hr) increasing further at 2.5 hr. Most of the radioactivity in the alkoxy diacylglycerols was found in the 1-0-alkyl moiety.     

Incorporation of [1-14C] acetate into lipids of soybean cell suspensions

Suspension cultures of soybean cells incorporated [1-¹⁴C] acetate very rapidly into the fatty acid moieties of phospholipids and glycolipids when incubated at 26 C for up to 22 hr. The most rapidly labeled lipid was 3-sn-phosphatidylcholine, which contained 58% of the total fatty acid radioactivity after 16 min; more than 75% of this label was found to be in the oleic acid of the phosphatidylcholine. After longer periods of incubation, the proportion of¹⁴C label decreased exponentially in phosphatidylcholine and increased markedly in an unidentified phospholipid (tentatively,bis-phosphatidic acid), di- and triacylglycerols, and glycolipids. The proportion of radioactivity in oleic acid also decreased exponentially, accompanied by increases in linoleic acid first and then in linolenic acid. Most of the labeled linolenic acid at 22 hr was found in the unidentified phospholipid, di- and triacylglycerols, and the glycolipid fraction. Contribution no. 537, Ottawa Research Station, Agriculture Canada. A preliminary report was presented at the 20th International Conference on the Biochemistry of Lipids at Aberdeen, Scotland, September 1977.     

Metabolism of actinic skin tumors: Incorporation of14C-acetate into lipids

Biochemical parameters of normal and actinically induced tumorous skin were compared. Similar respiratory rates and respiratory quotients were observed. However, both quantitative and qualitative differences occur in these tissue's ability to incorporate¹⁴C-acetate into lipids.     

Metabolism of 1,2-(1-14C) dipalmitoyl phosphatidylcholine in the developing brain

Thirteen-day-old rats were divided into two groups; one group received 1,2-(1-¹⁴C) dipalmitoyl phosphatidylcholine and the other 1-¹⁴C palmitic acid in the form of an intraperitoneal injection. One half of the total number of rats in each group was sacrificed 24 hr after injection, and the other half was allowed to survive for 17 days after the injection. Radioactivity incorporated into brain and liver total lipids and into individiual polar lipid components of the brain was determined at both intervals. Phosphatidylcholine was isolated and partially deacylated with phospholipase A₂ fromCrotalus Admanteus venom. The ratio of radioactivity FA 2/FA 1 (fatty acid attached to 2 and 1 carbon of the glycerol moiety) 24 hr after the injection was 8.3, when the tracer was radioactive phosphatidylcholine, compared to only 0.7 when radioactive palmitate was injected. From this different labeling ratio and different pattern of labeling the polar lipid components, it was concluded that the radioactive phosphatidylcholine was not deacylated completely before being taken up directly into the brain. Possibilities are discussed to show that the observed radioactive ratio could result from direct uptake of intact phosphatidylcholine, with little or no restriction from the blood brain barrier system, followed by partial degradation by phospholipase A₁ in the brain itself. Presented in part at the AOCS Meeting, New Orleans, April 1973.     

Incorporation of [1-14C]-oleic acid into neutral glycerides and phosphoglycerides of mouse brain

The incorporation of [1-¹⁴C]-oleic acid into the neutral glycerides and phosphoglycerides of adult mouse brain was examined between 1 and 80 min after intracerebral injection. Radioactivity of the free oleic acid in brain decreased rapidly with a half-life of ca. 5 min. The specific radioactivity of the phosphatidic acids was highest at 1 min after injection. This was followed by the diacylglycerols and triacylglycerols which attained a maximum specific radioactivity at 3 and 20 min after injection, respectively. Specific radioactivities of the neutral glycerides were higher than the phosphoglycerides. A larger proportion of the radioactivity in the diacylglycerols was transferred to the phosphoglycerides than to the triacylglycerols. Among the phosphoglycerides, radioactivity was actively incorporated into the inositol phosphoglycerides. The specific radioactivity of the inositol phosphoglycerides was higher than the diacylsn-glycero-3-phosphorylcholines, and the kinetics of incorporation of radioactivity into these lipids was also different. A water soluble material was found which showed maximum specific radioactivity at 6–10 min after injection. The properties of this water soluble material suggested that it may be an intermediate involved in the acyl group metabolism of phosphoglycerides in brain.     

Turnover of label from [1-14C] linolenic acid in phospholipids of coho salmon,Oncorhynchus kisutch

Juvenile coho salmon were injected intraperitoneally with [1-¹⁴C] linolenic acid, and sampled at 24, 120, and 240 hr. Liver, heart, and gill lipids were extracted, analyzed, and halflives of individual liver glycerophospholipids and n-3 fatty acids determined from rates of loss of radioactivity. Incorporation of label into gill was much less than into either heart or liver. Total acyl halflife was shorter for the choline phospholipids than for the ethanolamine phospholipids, as were the halflives of all individual n-3 fatty acids. Eicosapentaenoic acid (20∶5n-3) had the shortest halflife in both phospholipids (50–60 hr), while docosapentaenoic acid (22∶5n-3) and docosahexaenoic acid (22∶6n-3) had much longer halflives. Specific activities of the shorter chain n-3 fatty acids were much greater than the longer, more unsaturated homologs at all times, suggesting possible differences in their mechanisms of incorporation into phospholipids. Diacylglycerol analysis indicated that de novo synthesis could be responsible for the incorporation of only a small portion of the labeled long chain fatty acids found in phospholipids. The fatty acid halflives reported here for salmon are in general agreement with those found previously in mammals. Technical Paper No. 5238, Oregon Agricultural Experiment Station, Oregon State University, Corvallis, Oregon 97331. This material is taken in part from a thesis submitted in partial fulfillment of the requirements for the MS degree in 1978.     

Incorporation of [1-14C] acetate into phospholipids by soybean seedlings

Incorporation of [1-¹⁴C] acetate into lipids by slices of soybean seedlings was studied. The results were as follows: (a) the greatest amount of radioactivity was detected in the phospholipid fraction prepared from the main axis; (b) in the cotyledons, radioactivity was about the same in pigment, phosphatidylethanolamine, and phosphatidylglycerol; (c) phosphatidylcholine was the main phospholipid labeled in the axis; (d) the distribution of radioactive fatty acids in the axis suggested that this tissue has the capacity for both phospholipid synthesis and fatty acid desaturation.     

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.     

Effects of dietary linolenate on the fatty acid composition of brain lipids in rats

Weanling male rats were fed hydrogenated coconut oil to induce essential fatty acid (EFA) deficiency. After 15 weeks, the rats were divided into six groups. Five groups were fed graded amounts of purified linolenate (18∶3ω3) with a constant amount of linoleate (18∶2ω6) for six weeks. Fatty acid composition was determined in brain lipids. Increasing dietary 18∶3ω3 resulted in a decrease in arachidonic acid (20∶4ω6), docosatetraenoic acid (22∶4ω6) and docosapentaenoic acid (22∶5ω6), whereas 18∶2ω6 and eicosatrienoic acid (20∶3ω6) were increased both in total lipids and phospholipids. These results suggest that dietary 18∶3ω3 exerts its inhibitory effect mainly on the desaturation of 20∶ω6 to 20∶4ω6 in brain lipids. Linolenate was undetectable in brain lipids from any dietary treatments. The levels of eicosapentaenoic acid (20∶5ω3) in groups receiving dietary 18∶3ω3 were not different from that of the group receiving no 18∶3ω3. These results indicate that, in the brain, 18∶3ω3 is rapidly converted mainly to 22∶6ω3 without being accumulated and imply that dietary 18∶3ω3 can modulate the level of precursor of diene prostaglandins (PG) but not that of triene PG in the rat brain.     

Metabolism of [1-14C]docosahexaenoate (22∶6n−3), [1-14C]eicosapentaenoate (20∶5n−3) and [1-14C]linolenate (18∶3n−3) in brain cells from juvenile turbotScophthalmus maximus

The incorporation of [1-¹⁴C]18∶3n−3, (LNA) and [1-¹⁴C]-22∶6n−3 (DHA), and the metabolismvia the desaturase/elongase pathways of [1-¹⁴C]LNA, and [1-¹⁴C]20∶5n−3 (EPA) were studied in brain cells from newly-weaned (1-month-old) and 4-month-old turbot. The rank order of the extent of net incorporation of both LNA and DHA into glycerophospholipids was total diradyl glycerophosphocholines (CPL)> total diradyl glycerophosphoethanolamines (EPL)> phosphatidylserine (PS) and phosphatidylinositol (PI) and was independent of the polyunsaturated fatty acid added, the age of the fish and the time of incubation. However, the rate of incorporation of LNA into total lipid, CPL, EPL and PS was significantly greater than the rate of incorporation of DHA, and there was a significantly greater amount of DHA incorporated into EPL than LNA. There was no significant difference between the amounts of LNA and DHA incorporated into total lipid, CPL, PS and PI. Therefore, little preferential uptake and incorporation of DHA into brain cells was apparent. In 24-h incubations, on average 1.1% and 8.5% of radioactivity from [1-¹⁴C]LNA and [1-¹⁴C]EPA, respectively, were recovered in the DHA fraction. Therefore, LNA cannot contribute significantly to brain DHA levels in the turbot but EPA can. There were no significant differences between the amounts of radioactivity from either [1-¹⁴C]LNA or [1-¹⁴C]EPA recovered in the individual products/intermediates of the desaturase pathways in brain cells from 30-day-old and 120-day-old turbot.     

Incorporation of 1-14C-Acetate into C26 fatty acids of the marine spongeMicrociona prolifera

The incorporation of 1-¹⁴C-acetate into the many fatty acids of the marine spongeMicrociona prolifera was investigated. Probable precursors of 26∶2Δ5,9 and 26∶3Δ5,9,19 showed high levels of radioactivity, supporting the following pathways for the biosynthesis of C₂₆ acids: $$\begin{array}{*{20}c} {16:0 \to \to \to 26:0 \to 26:1\Delta 9 \to 26:2\Delta 5,9} \\ {16:1\Delta 9 \to \to \to 26:1\Delta 19 \to } \\ {26:2\Delta 9,19 \to 26:3\Delta 5,9,19} \\ \end{array}$$ Degradation of the unsaturated C₂₆ acids at their double bonds showed that the¹⁴C was concentrated near the carboxyl end of the chain. Hence, chain elongation was the major mechanism for acetate incorporation into these acids.     

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.     

Intravenously injected [1-14C]arachidonic acid targets phospholipids, and [1-14C]palmitic acid targets neutral lipids in hearts of awake rats

The differential uptake and targeting of intravenously infused [1-¹⁴C]palmitic ([1-¹⁴C] 16∶0) and [1-¹⁴C]arachidonic ([1-¹⁴C]20∶4n−6) acids into heart lipid pools were determined in awake adult male rats. The fatty acid tracers were infused (170 μCi/kg) through the femoral vein at a constant rate of 0.4 mL/min over 5 min. At 10 min postinfusion, the rats were killed using pentobarbital. The hearts were rapidly removed, washed free of exogenous blood, and frozen in dry ice. Arterial blood was withdrawn over the course of the experiment to determine plasma radiotracer levels. Lipids were extracted from heart tissue using a two-phase system, and total radioactivity was measured in the nonvolatile aqueous and organic fractions. Both fatty acid tracers had similar plasma curves, but were differentially distributed into heart lipid compartments. The extent of [1-¹⁴C]20∶4n−6 esterification into heart phospholipids, primarily choline glycerophospholipids, was elevated 3.5-fold compared to [1-¹⁴C]16∶0. The unilateral incorporation coefficient, k ^*, which represents tissue radioactivity divided by the integrated plasma radioactivity for heart phospholipid, was sevenfold greater for [1-¹⁴C]20∶4n−6 than for [1-¹⁴C]16∶0. In contrast, [1-¹⁴C]16∶0 was esterified mainly into heart neutral lipids, primarily triacylglycerols (TG), and was also found in the nonvolatile aqueous compartment. Thus, in rat heart, [1-¹⁴C]20∶4n−6 was primarily targeted for esterification into phospholipids, while [1-¹⁴C]16∶0 was targeted for esterification into TG or metabolized into nonvolatile aqueous components.     

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.     

Incorporation of linolenic-1-C14 acid into eicosapentaenoic and docosahexaenoic acids in fish

Following intraperitoneal injection of methyl linolenate-1-C¹⁴ into kelp bass,Paralablax clathratus, the highly polyunsaturated fatty acids of their body fats were concentrated by low temperature crystallization from acetone, and eicosapentaenoic and docosahexaenoic acids were isolated from the concentrate by reversed-phase chromatography and hydrogenated. The resulting arachidic and behenic acids were degraded stepwise to margaric acid, and the distribution of activity was determined. The results indicate that the injected linolenic acid was converted to eicosapentaenoic acid and the latter incorporated into docosahexaenoic acid. A probable conversion pathway is linolenic acid→6,9,12,15-octadecatetraenoic acid→8,11,14,17-eicosatetraenoic acid→5,8,11,14,17-eicosapentaenoic acid→7,10,13,16,-19-docosapentaenoic acid→4,7,10,13,16,19-docosahexaenoic acid. Supported by a training grant from the National Heart Institute, Bethesda, Md. This paper based partially on work performed under Contract AT(04-1)GEN-12 between the Atomic Energy Commission and the University of California.     

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.     

High pressure liquid chromatography of autoxidized lipids: II. Hydroperoxy-cyclic peroxides and other secondary products from methyl linolenate

A previous study of autoxidation products by high pressure liquid chromatography (HPLC) of methyl oleate and linoleate was extended to methyl linolenate. Autoxidized methyl linolenate was fractionated by HPLC either after reduction to allylic alcohols on a reverse phase system, or directly on a micro silica column. Isolated oxidation products were characterized by thin layer and gas liquid chromatography and by ultraviolet, infrared, nuclear magnetic resonance and mass spectrometry. Secondary products from the autoxidation mixtures (containing 3.5–8.5% monohydroperoxides) included epoxy unsaturated compounds (0.2–0.3%), hydroxy or hydroperoxy-cyclic peroxides (3.8–7.7%), epoxy-hydroxy dienes (<0.1%), dihydroxy or dihydroperoxides with conjugated diene-triene and conjugated triene systems (0.9–2.9%). Cyclization of the 12- and 13-hydroperoxides of linolenate would account for their lower relative concentration than the 9- and 16-hydroperoxides. Dihydroperoxides may be derived from the 9- and 16-linolenate hydroperoxides. Cyclic peroxides and dihydroperoxides are suggested as important flavor precursors in oxidized fats.