Metabolic transformation of intracraneally injected [1-14C]linoleic and [1-14C]α-linolenic acids in malnourished developing rats

The effect of a low protein diet during pregnancy and lactation on the fatty acid composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from brains of ten-day-old rats was studied. The results indicated that partial deprivation of protein during early development was associated with an increase in the fatty acids of the n−9 family in PC. The fatty acids of the linoleic acid series decreased in PE but were not modified in PC. These minor changes did not affect the double bond index values either in PC or in PE. The effect of protein depletion on thein vivo metabolic transformation of intracraneally injected [1-¹⁴C]linoleic and [1-¹⁴C]α-linolenic acids was also studied. The percentage distribution of the labeled precursors and their derivatives among PC and PE differed from that of mass distribution. These results indicate that the direct uptake of polyunsaturated fatty acids from the blood and/or the low turnover rate of these acids incorporated into PC and PE might be involved in maintaining the fatty acid pattern of these brain lipids.     

Labeled oxidation products from [1-14C], [U-14C] and [16-14C]-palmitate in hepatocytes and mitochondria

When [1-¹⁴C], [U-¹⁴C], and [16-¹⁴C]palmitate were oxidized by isolated rat hepatocytes, there was a differential distribution of label as a percent of total oxidized products, such that¹⁴CO₂ from [1-¹⁴C]>[U-¹⁴C]>[16-¹⁴C]-palmitate and acid-soluble radioactivity from [16-¹⁴C]>[U-¹⁴C]>[1-¹⁴C]palmitate. The oxidation of [2,3-¹⁴C]succinate to¹⁴CO₂ by isolated hepatocytes was only 9.1% of that from [1,4-¹⁴C]succinate, demonstrating that the differences in distribution of labeled products are in part due to less¹⁴CO₂ production from label in the even carbon positions entering the citric acid cycle. Apparent total ketone body production from [16-¹⁴C]palmitate was markedly higher than [1-¹⁴C], and [U-¹⁴C]palmitate. In addition, the¹⁴C-acetone:¹⁴CO₂ ratio derived from decarboxylation of labeled acetoacetate from [1-¹⁴C]palmitate was less than 1 and positively correlated to the rate of fatty acid oxidation in hepatocytes. These findings indicate that the known preferential incorporation of the omega-C₂ unit of fatty acids into¹⁴C-ketone bodies also contributed to the differential distribution of labeled products and that this contribution was greatest at the lower rates of fatty acid oxidation. In isolated mitochondria, the distribution of label to¹⁴CO₂ and acid-soluble radioactivity from [1-¹⁴C], [U-¹⁴C] and [16-¹⁴C]palmitate was qualitatively similar to that seen with hepatocytes. The distribution of label from [1-¹⁴C]acetylcarnitine to¹⁴CO₂ and¹⁴C-ketone bodies by mitochondria was identical to that observed from [1-¹⁴C]palmitate, indicating that the higher rates of¹⁴CO₂ production from [1-¹⁴C]palmitate cannot be explained by a preferential oxidation in the citric acid cycle of either extramitochondrial acetyl-CoA (generated in peroxisomes) or the carboxyl terminal of the fatty acid. As shown by others in cell-free systems, we observed that the total oxidation of [16-¹⁴C]palmitate by hepatocytes and mitochondria was significantly less than [1-¹⁴C] and [U-¹⁴C]palmitate, suggesting either incomplete mitochondrial β-oxidation or incomplete degradation of peroxisomal oxidation products. The data indicate that this incomplete oxidation does not, however, contribute to the differential distribution of label to oxidized products.     

Hepatic metabolism of 1-14C octanoic and 1-14C margaric acids

The hepatic metabolism of 1-¹⁴C margaric acid, a 17 carbon long chain saturated fatty acid which is present in the liver in trace amounts, was compared with 1-¹⁴C octanoic acid and 1-¹⁴C palmitic acid to determine if the enhanced oxidation of medium chain fatty acids to CO₂ was dependent on fatty acid chain length or the endogenous pool size of the fatty acid substrate. Despite the fact that endogenous margarate is present in trace amounts, there was no significant difference in the oxidation of margarate and palmitate to CO₂, while the oxidation of octanoate to CO₂ was significantly more rapid. Both margarate and palmitate were more readily incorporated into lipid soluble products in contrast to the low rate of incorporation of octanoate. However, margarate was less readily incorporated into triglyceride, phospholipid and monoglyceride than palmitate. These studies suggest that the chain length rather than hepatic content of the fatty acid determines whether the carboxyl group of equimolar amounts of a 1-¹⁴C-carboxyl labeled fatty acid will be preferentially oxidized to CO₂ or incorporated into tissue lipid in the liver.     

Hepatic metabolism of 1-14C octanoic and 1-14C palmitic acids

The hepatic metabolism of 1⁻¹⁴C octanoic acid was compared with that of 1⁻¹⁴C palmitic acid in male rats which were fed. After intraportal injection only 1/6 to 1/18 as much octanoic acid as palmitic acid was incorporated into hepatic lipids. In contrast, octanoic acid yielded two to four times as much water-soluble product as did palmitic acid. Similar, but even more impressive, differences between the incorporation of these fatty acids into hepatic lipids were observed in liver slices incubated with¹⁴C octanoate and¹⁴C palmitate. The oxidation of octanoate to CO₂ was more than 10 times as great as that of palmitate. With both substrates, triglycerides comprised almost half the labeled lipid recovered. However octanoate yielded a higher proportion of labeled, unesterified fatty acids and a lower proportion of labeled phospholipid and monoglycerides than did palmitate. Most of the¹⁴C recovered in hepatic lipids after incubation with 1⁻¹⁴C octanoate was found in the carboxyl groups of long-chain fatty acids, suggesting that the latter had been synthesized from 2-carbon fragments formed from the oxidation of octanoate. In contrast, only a small fraction of the palmitate was elongated. The similarities and differences between the metabolism of octanoic and palmitic acid in liver and intestine, and the possible nutritional significance of octanoic acid are discussed.     

Partial synthesis of [1-14C]phytanic acid

[1-¹⁴C] Phytanic acid has been prepared in good yield from the unlabeled acid. Pristanyl iodide, prepared from the latter by a modified Hunsdieker reaction, is converted to the corresponding [¹⁴C]-nitrile by reaction with sodium [¹⁴C] cyanide in dimethyl sulphoxide; hydrolysis of the [¹⁴C] nitrile yields [1-¹⁴C]phytanic acid. The labeled acid should prove to be a useful substrate for the diagnosis of Refsum's disease.     

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.     

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 [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.     

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.     

Differential biohydrogenation and isomerization of [U-13C]Oleic and [1-13C]Oleic acids by mixed ruminal microbes

The additional mass associated with ¹³C in metabolic tracers may interfere with their metabolism. The comparative isomerization and biohydrogenation of oleic, [1-¹³C]oleic, and [U-¹³C]oleic acids by mixed ruminal microbes was used to evaluate this effect. The percent of stearic, cis-14 and- 15, and trans-9 to-16 18∶1 originating from oleic acid was decreased for [U-¹³C]oleic acid compared with [1-¹³C]oleic acid. Conversely, microbial utilization of [U-¹³C]oleic acid resulted in more of the ¹³C label in cis-9 18∶1 compared with [1-¹³C]oleic acid (53.7 vs. 40.1%). The isomerization and biohydrogenation of oleic acid by ruminal microbes is affected by the mass of the labeled tracer.     

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.     

Aromatization of [1-14C]heptane over “nonacidic” platinum-alumina catalyst

[1-¹⁴C]Heptane was aromatized under 5 atm pressure over a “nonacidic” 3% platinum on alumina catalyst maintained at 500 °C. The reactant was pumped in at a HLSV of 0.88, and the hydrogen-to-heptane mole ratio was 5.1. The samples of product were collected (one during the first 90 min and another during the next 90 min), the toluene was separated from the respective samples, and the radioactivity in each position of the toluene was determined by a degradation sequence. The distribution of radioactivity in sample 1 was as follows: in position 1–2.6%, 2–38.6%; 3–14.5%; 4–4.2%, and in the methyl group—40.1%. Approximately similar distribution of radioactivity was found in sample 2. 1,6-Ring closure is the most important pathway leading to the formation of toluene from [1-¹⁴C]heptane; lesser amounts of repetitive, 1,5-ring closure and hydrogenolysis, and bond shift reactions followed by 1,6-ring closure and dehydration can explain the distribution of radioactivity. The participation of 1,7-ring closure in the reaction is also indicated.     

Incorporation of [1-14C] acetate into fatty acids of the crustaceansDaphnia magna andCyclops strenus in relation to temperature

Daphnia magna andCyclops strenus were maintained in aquaria containing sodium [1-¹⁴C] acetate and the effect of temperature on labeling of their lipids was investigated. Incorporation of radioactivity in total lipids was slowed by a factor of 4 in cold-exposed (5C) specimens compared to those incubated at 25 C. There was no significant difference in the distribution of label in the lipid classes of animals incubated at the two extreme temperatures. Decrease of the temperature from 25 to 5 C brought about a considerable reduction in the formation of palmitic and stearic acids and an increase in labeling of monounsaturated (18∶1) fatty acids inD. magna. Docosapolyenoic acids were absent from lipids of this crustacean.C. strenus directed a higher proportion of radioactivity into both oleic and docosahexaenoic acids upon cold exposure. In response to decrease of the temperature,D. magna formed a less unsaturated fatty acid population, as judged from dpm ratios of total saturated to total unsaturated fatty acids, thanC. strenus. Inability to form and accumulate docosapolyenoic fatty acids byD. magna might be related to their poor survival at reduced temperatures.     

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.     

3-[3-(1-哌啶甲基)苯氧基]丙胺的合成工艺改进

以间羟基苯甲醛为起始原料，经由氧烷基化、缩合、水解得3-[3-(1-哌啶甲基)苯氧基]丙胺，收率49．1％，结构经NMR确证。     

Synthesis and characterization of [1-13C]-and d8-arachidonic acid

Methyld ₈- and [1-¹³C] 5,8,11,14-eicosatetraenoate (arachidonate) were prepared from a common synthetic precursor, 4,7,10,13-nonadecatetrayn-1-ol. The purified products were characterized by gas chromatography-mass spectrometry. Mass spectra oft-butyldimethylsilyl esters ofd ₈-and [1-¹³C]-arachidonic acid showed a most intense [M-57]⁺ peak at high mass. The isotopic purity of methyl [1-¹³C] arachidonate was 99% and that of methyld ₈-arachidonate was 56%. Whend ₈-arachidonic acid was prepared by direct deuteration of 5,8,11,14-eicosatetraynoic acid, the isotopic purity of the sample was 86%.     

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.     

Lipid products formed during desaturation of [1-14C] stearyl CoA by hen liver microsomes

A number of lipid products are formed during the desaturation of stearyl CoA by hen liver microsomes. This article presents an analysis of the products formed when [1-¹⁴C] stearyl CoA is incubated with hen liver microsomes for various time periods. [1-¹⁴C] Oleyl CoA was the first radioactive unsaturated product formed. Synthesis of phospholipids containing [1-¹⁴C] oleic acid occurs only after the desaturase activity has begun to decline. The specific radioactivity of [1-¹⁴C] oleyl CoA was similar to the specific radioactivity of [1-¹⁴C] stearyl CoA at all time periods tested. The specific radioactivities of [1-¹⁴C] oleic acid and phospholipids containing [1-¹⁴C] oleic acid were much lower than that of the [1-¹⁴C] stearyl CoA.