Synthesis of ethyl arachidonate-19,19,20,20-d 4 and ethyl dihomo-γ-linolenate-19,19,20,20-d 4

Ethyl 5,8,11,14-eicosatetraenoate-19,19,20,20-d ₄ and ethyl 8,11,14-eicosatrienoate-19,19,20,20-d ₄ were synthesized by Grignard coupling of the methanesulfonyl ester of 2,5-undecadiyn-1-ol-10,10,11,11-d ₄ with 5,8-nonadiynoic acid and 8-nonynoic acid, respectively. The coupled products upon Lindlar reduction, followed by the preparation of their ethyl esters, yielded deuteriated ethyl arachidonate and ethyl dihomo-γ-linolenate, which were completely characterized by¹³C and¹H nuclear magnetic resonance and mass spectral analysis.     

Biosynthesis of hydrocarbons by algae: Decarboxylation of stearic acid to N-heptadecane inAnacystis nidulans determined by13C- and2H-labeling and13C nuclear magnetic resonance

The distribution of isotopic labels inn-heptadecane enriched from [1,2-¹³C] and [2-¹³C, 2-²H₃) acetates byAnacystis nidulans has been determined by¹³C nuclear magnetic resonance (¹³C NMR). Labeling with [1,2-¹³C] acetate is consistent with assembly from¹³C−¹³C units derived from an acetate “starter” group and 8 malonate units, as in fatty acid biosynthesis, followed by production of a methyl group through bond cleavage of the terminal¹³C−¹³C unit. A comparison of the hydrocarbon with palmitic acid (the only fatty acid produced in sufficient amount for NMR analysis) enriched from [2-¹³C,2-²H₃]acetate by the same culture shows that they have retained the same fraction of²H at corresponding sites, and have therefore undergone identical biosynthetic and hydrogen-deuterium exchange processes, as would be expected ifn-heptadecane originates from de novo-synthesized stearic acid. NRCC No. 18251.     

The questionable role of a microsomal °8 acyl-CoA-dependent desaturase in the biosynthesis of polyunsaturated fatty acids

Several experimental approaches were used to determine whether rat liver and testes express an acyl-CoA-dependent δ8 desaturase. When [1-¹⁴C]5, 11, 14-eicosatrienoic acid was injected via the tail vein, or directly into testes, it was incorporated into liver and testes phospholipids, but it was not metabolized to other labeled fatty acids. When [1-¹⁴C]11, 14-eicosadienoic acid was injected, via the tail vein or directly into testes, or incubated with microsomes from both tissues, it was only metabolized to 5,11, 14-eicosatrienoic acid. When ethyl 5,5,11,11,14,14-d₆-5,11,14-eicosatrienoate was fed to rats maintained on a diet devoid of fat, it primarily replaced esteri-fied 5,8,11-eicosatrienoic acid, but not arachidonic acid. No labeled linoleate or arachidonate were detected. Dietary ethyl linoleate and ethyl 19,19,20,20-d₄-1,2-¹³C-11,14-eicosadienoate were about equally effective as precursors of esterified arachidonate. The doubly labeled 11,14-eicosadienoate was metabolized primarily by conversion to 17,17,18,18-d₄-9,12-ocatdeca-dienoic acid, followed by its conversion to yield esterified arachidonate, with a mass four units greater than endogenous arachidonate. In addition, the doubly labeled substrate gave rise to a small amount of arachidonate, six mass units greater than endogenous arachidonate. No evidence was obtained, with the radiolabeled substrates, for the presence of a δ8 desaturase. However, the presence of an ion, six mass units greater than endogenous arachidonate when doubly labeled 11, 14-eicosa-dienoate was fed, suggests that a small amount of the substrate may have been metabolized by the sequential use of δ8 and δ5 desaturases.     

Stimulation of hepatic lipogenesis by eicosa-5,8,11,14-tetraynoic acid in mice fed a high linoleate diet

Liver slices, from mice fasted for one day and then refed for three days either a 15% corn oil diet or a 15% corn oil diet containing eicosa-5,8,11,14-tetraynoic acid (TYA), were incubated with [1-¹⁴C] acetate or [³H]H₂O to determine lipogenic capacity. Dietary TYA produced a twofold stimulation in fatty acid and cholesterol synthesis. TYA also caused an increase in the relative proportion of linoleate (C_18∶2) and a decrease in that of arachidonate (C_20∶4) in liver. Thus, (a) despite high levels of C_18∶2, hepatic lipogenesis can be increased, and (b) even short term feeding of TYA can alter the hepatic fatty acid composition presumably by inhibition of arachidonate synthesis from linoleate.     

Trans fatty acids: Positional specificity in brain lecithin

Fifteen-day-old rats were divided into three groups: one group received an intracerebral injection of 5 μ Ci of 9-trans, 12-trans [1-¹⁴C] octadecadienoic acid; the second group was given 5 μCi of the same compound plus an equal wt of nonradioactive allcis arachidonic acid; the third group was given 5 μCi of 9-trans [1-¹⁴C] octadecenoic acid. All animals were sacrificed 8 hr after injection. Glycerophosphocholine (GPC) was isolated and partically deacylated with phospholipase A₂ fromCrotalus Adamanteus venom. The results of this study were as follows: 1) aftert [1-¹⁴C] 18∶1 injection, there was twice as much radioactivity in the 1-position as in the 2-position; 2) whentt [1-¹⁴C] 18∶2 was injected, more than 90% of the total radioactivity was found in the 2-position; 3) followingtt[1-¹⁴C]-18∶2 +nonradioactive arachidonate injection, ca. 75% of the total radioactivity still remained in the 2-position; and 4) all of the injected [1-¹⁴C]-tracers showed evidence of undergoing β-oxidation to form acetyl-CoA, which was converted to radioactive palmitate. The possibility is discussed that the observed distribution pattern of the injected radioactive tracers may be attributed to tissue metabolic specificity. Ramifications of the deposition of dietarytrans fatty acids in the brain during the developmental stage of the central nervous system are also discussed.     

[3-13C] γ-linolenic acid: A new probe for 13C nuclear magnetic resonance studies of arachidonic acid synthesis in the suckling rat

Our objective was to develop a suitable probe to study metabolism of polyunsaturated fatty acids by ¹³C nuclear magnetic resonance (NMR) in the suckling rat pup. [3-¹³C] γ-Linolenic acid was chemically synthesized, and a 20 mg (Experiment 1) or 5 mg (Experiment 2) dose was injected into the stomachs of 6–10-day-old suckling rat pups that were then killed over a 192 h (8 d) time course. ¹³C NMR showed that ¹³C in γ-linolenate peaked in liver total lipids by 12-h post-dosing and that [5-¹³C]-arachidonic acid peaked in both brain and liver total lipids 48–96 h post-dosing. ¹³C enrichment in brain γ-linolenic acid was not detected by NMR, but gas chromatography-combustion-isotope ratio mass spectrometry showed that its mass enrichment in brain phospholipids at 48–96 h post-dosing was 1–2% of that in brain arachidonic acid. ¹³C was present in liver and brain cholesterol and in perchloric acid-extractable water-soluble metabolites in the brain, liver and carcass. We conclude that low but measurable amounts of exogenous γ-linolenic acid do access the suckling rat brain in vivo. The slow time course of [5-¹³C] arachidonic acid appearance in the brain suggests most of it was probably transported there after synthesis elsewhere, probably in the liver. Some carbon from γ-linolenic acid is also incorporated into lipid products other than n−6 long-chain polyunsaturated fatty acids.     

Biosynthesis of [14C]arachidonic acid from [14C]linoleate in primary cultures of rat Sertoli cells

The conversion of [¹⁴C]linoleate to [¹⁴C]arachidonate by rat Sertoli cells was established by use of primary cultures. Most of the¹⁴C from [1-¹⁴C]linoleate was located in C-3 of the synthesized arachidonate, indicating that the labeled tetraene had originated largely by elongation and desaturation of the intact labeled substrate rather than by mere addition of¹⁴C-acetate generated by bio-oxidation of the radioactive substrate to an already existing 18-carbon precursor. Although a relatively small amount of¹⁴C was present in 18∶3ω6 and a relatively large amount of¹⁴C was present in 20∶2, it was not possible from these data to establish the relative importance of 20∶2 in the biosynthesis of arachidonic acid in rat Sertoli cells.     

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.     

The gastrointestinal handling and metabolism of [1-13C]palmitic acid in healthy women

The gastrointestinal handling and metabolism of [1-¹³C]palmitic acid given as the free fatty acid was examined in six healthy women by measuring the excretion of¹³C-label in stool and in breath as¹³CO₂. The gastrointestinal handling of [1-¹³C]palmitic acid was compared with the apparent absorption of dietary lipid by measuring lipid losses in stool. The variation both within and between subjects was determined by repeating the study in the same individuals on separate occasions. The time course for excretion of label in stool over the five-day study period followed a common pattern, with most of the label excreted over the first two days of the stool collection.¹³C-Label excreted in stool over the five-day study period was 14.3±9.8% of that administered and on repeating the trial was 31.6±24.7% (not significantly different due to variability); there was poor agreement within subjects. Lipid excreted in stool expressed as a percentage of ingested lipid was 5.2±4.4% in Trial 1 and 5.9±4.0% in Trial 2, and was the same in each individual on repeating the trial. There was no clear relationship between the excretion of¹³C-label and lipid in stool (Trial 1:R=−0.43,P>0.40; Trial 2:R=−0.02,P>0.97). On the first occasion, 22.0±4.5% of the administered label was excreted on breath over the 15-h study period and on repeating the trial was 15.8±9.5% (not significantly different) with poor repeatability in a given individual. There was an inverse relationship between the proportion of¹³C-label excreted in stool and that excreted on breath in Trial 1 (R=−0.80,P>0.06) with a weaker association observed in Trial 2 (R=−0.49,P>0.32). Correcting for differences in the apparent absorption of label reduced the variability in its excretion in breath observed between subjects, particularly in Trial 2. It is concluded that although there are differences in the gastrointestinal handling of [1-¹³C]palmitic acid both within and between healthy adults, the postprandial oxidation of absorbed substrate was similar. The assumptions underlying these observations need to be examined by characterizing the nature of¹³C-label in stool.     

Esterification of 8,11,14-eicosatrienoate and arachidonate into alkylacyl- and diacylglycerophosphocholine by vascular endothelial cells

Agonist-stimulated phospholipases release arachidonate, but not 8,11,14-eicosatrienoate, from human endothelial cells. One source of the arachidonic acid is deacylation of 1-alkyl-2-arachidonoyl-glycerophosphocholine, with subsequent conversion of some of the resultant lysophospholipid to platelet-activating factor. This study has compared the distribution of incorporated 8,11,14-[¹⁴C]-eicosatrienoate in alkylacyl-GPC and diacyl-GPC with that of [¹⁴C]arachidonate synthesized endogenously by desaturation of the 8,11,14-[¹⁴C]eicosatrienoate. Cells were incubated for 24 or 48 hr with 8,11,14-[¹⁴C]eicosatrienoate, and the resultant mixture of¹⁴C-fatty acids in the cellular lipids was characterized by gas chromatography. The choline phospholipids were then separated, hydrolyzed with phospholipase C and derivatized to diradylbenzoates. Gas chromatographic analysis indicated extensive incorporation of [¹⁴C]eicosatrienoate, as well as [¹⁴C]arachidonate, into alkylacyl-GPC. Although the ratio of esterified [¹⁴C]arachidonate to [¹⁴C]eicosatrienoate was greater in alkylacyl-GPC than in diacyl-GPC, the enrichment with [¹⁴C]arachidonate was far less than the ratio of arachidonate/eicosatrienoate released from these cells. These results thus support the hypothesis that the acyl specificity of polyunsaturated fatty acid release is provided by the agonist-stimulated phospholipase A₂ rather than the composition of the alkylacyl-GPC.     

Regulation of the metabolism of linoleic acid to arachidonic acid in rat hepatocytes

When 5×10⁶ hepatocytes were incubated for 40 min with from 0.15 to 0.60 mM [1-¹⁴C]linoleic acid, [1-¹⁴C]6,9,12-octadecatrienoic acid, or [1-¹⁴C]8,11,14-eicosatrienoic acid, there was a concentration-dependent acylation of radioactive metabolites into both triglycerides and phospholipids. When the concentration of either [1-¹⁴C]linoleic acid or [1-¹⁴C]8,11,14-eicosatrienoic acid exceeded 0.3 mM, there was no further increase in the metabolism of either fatty acid to other (n−6) metabolites. When the concentration of [1-¹⁴C]6,9,12-octadecatrienoic acid exceeded 0.15 mM, there was an apparent substrate-induced inhibition in its metabolism to 8,11,14-eicosatrienoic acid. With all three substrates (0.3 mM), there was time-dependent metabolism to other (n−6) acids. Cells then were incubated simultaneously with 0.3 mM [1-¹⁴C]linoleic acid along with 0.15 to 0.45 mM 6,9,12-octadecatrienoic acid or 8,11,14-eicosatrienoic acid. These exogenous nonradioactive (n−6) acids suppressed but did not abolish the conversion of [1-¹⁴C]linoleate to radioactive arachidonate. These findings suggest that some linoleate is converted to arachidonate without intracellular mixing of 6,8,12-octadecatrienoic or 8,11,14-eicosatrienoic acids. This hypothesis is supported by the finding that exogenous linoleate did not markedly affect the metabolism of [1-¹⁴C]6,9,12-octadecatrienoic or [1-¹⁴C]8,11,14-eicosatrienoic acid by microsomal chain elongating or desaturating enzymes.     

Metabolism of long-chain fatty acids,alcohols and alkylglycerols in the fish parasiteParatenuisentis ambiguus (Acanthocephala)

Specific differences between the acyl composition of lipids on the helminthParatenuisentis ambiguus and its host eel, as shown previously, prompted us to study the lipid metabolism in this intestinal fish parasite. Adults and larvae ofP. ambiguus were fed various lipid precursors, e.g., fatty acids, long-chain alcohols and 1-O-alkylglycerols, which may occur as common nutrients of intestinal parasites. Incorporation of [1-¹⁴C]palmitic acid into neutral and polar lipids was found to be similar under aerobic and near-anaerobic conditions. In adult parasites maintained in culture medium supplemented with glucose, [1-¹⁴C]palmitic acid was incorporated mainly into triacylglycerols and phosphatidylcholines, whereas [1-¹⁴C]oleic acid was incorporated preferentially into triacylglycerols. In fasted adults, as well as in larvae, [1-¹⁴C]oleic acid was mainly transferred to phosphatidylcholines. Lipolytic activity was detected in adult parasites that had been incubated with radioactive trioleoylglycerol. [1-¹⁴C]Hexadecan-1-ol was oxidized inP. ambiguus at a high rate to labeled palmitic acid, which was incorporated into various lipid classes ofP. ambiguus. Small but significant proportions of radioactivity from hexadecan-1-ol were incorporated into ether glycerolipids of the parasite. A more direct precursor in ether glycerolipid metabolism, i.e.,rac-1-O-[1′-¹⁴C] hexadecylglycerol, was incorporated into alkyl and 1′-alkenyl moieties of choline and etha-nolamine etherglycerophospholipids ofP. ambiguus in high yield. High proportions of labeled diacylglycerols, triacylglycerols and steryl esters were detected in surface lipids as well as lipid extracts of the culture media after incubation ofP. ambiguus with [1-¹⁴C]palmitic or [1-¹⁴C]oleic acids. The results suggest that palmitic acid and oleic acid are incorporated into neutral and polar lipids ofP. ambiguus maintained in glucose medium quite differently with oleic acid showing a strong preference for triacylglycerols. However, the incorporation of palmitic acid in glucose-fed parasites was similar to that of oleic acid in fasted parasites, as well as in larvae. This may be explained by partial fatty acid depletion in fasted worms and rapid cell division in larvae, respectively.     

Methodology for the In Vivo Measurement of the Δ<Superscript>9</Superscript>-Desaturation of Myristic,Palmitic, and Stearic Acids in Lactating Dairy Cattle

There is limited methodology available to quantitatively assess the activity of the Δ⁹-desaturase enzyme in vivo without chemically inhibiting the enzyme or using radioactively labeled substrates. The objective of these experiments was to develop methodology to determine the incorporation and desaturation of ¹³C-labeled fatty acids into milk lipids. In a preliminary experiment, 3.7 g [1-¹³C]myristic acid ([1-¹³C]14:0), 19.5 g [1-¹³C]palmitic acid ([1-¹³C]16:0), 20.0 g [1-¹³C]stearic acid ([1-¹³C]18:0) were combined and infused into the duodenum of a cow over 24 h. In a following experiment, 5.0 g [1-¹³C]14:0, 40.0 g [1-¹³C]16:0, and 50.0 g [1-¹³C]18:0 were infused into the abomasums of separate cows as a bolus over 20 min or continuously over 24 h. Milk fat was extracted using chloroform:methanol. Fatty acids were methylated, and fatty acid methyl esters (FAME) were converted to dimethyl disulfide derivatives (DMDS). The FAME and DMDS were analyzed by gas chromatography mass spectrometry. In the preliminary experiment, ¹³C enrichment in 14:0 but not 16:0 or 18:0 was observed. When dosage amounts were increased in the following experiment, peak enrichments from the bolus infusion were observed at 8 h. Enrichments for continuous infusion peaked at 16 h for 14:0 and 18:0, and at 24 h for 16:0. The Δ⁹-desaturase products of these fatty acids were estimated to be 90% of cis-9 14:1, 50% of cis-9 16:1, and 59% of cis-9 18:1. This study demonstrates that ¹³C-labeled fatty acids may be utilized in vivo to measure the activity of the Δ⁹-desaturase enzyme.     

Transfer of arachidonate from phosphatidylcholine to phosphatidylethanolamine and triacylglycerol in guinea pig alveolar macrophages

Guinea pig alveolar macrophages were labeled by incubation with either arachidonate or linoleate. Arachidonate labeled phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triglycerides (TG) equally well, with each lipid containing about 30% of total cellular radioactivity. In comparison to arachidonate, linoleate was recovered significantly less in PE (7%) and more in TG (47%). To investigate whether redistributions of acyl chains among lipid classes took place, the macrophages were incubated with 1-acyl-2-[1-¹⁴C]arachidonoyl PC or 1-acyl-2-[1-¹⁴C]linoleoyl PC. After harvesting, the cells incubated with 1-acyl-2-[1-¹⁴C]linoleoyl PC contained 86% of the recovered cellular radioactivity in PC, with only small amounts of label being transferred to PE and TG (3 and 6%, respectively). More extensive redistributions were observed with arachidonate-labeled PC. In this case, only 60% of cellular radioactivity was still associated with PC, while 22 and 12%, respectively, had been transferred to PE and TG. Arachidonate transfer from PC to PE was unaffected by an excess of free arachidonate which inhibited this transfer to TG for over 90%, indicating that different mechanisms or arachidonoyl CoA pools were involved in the transfer of arachidonate from PC to PE and TG. Cells prelabeled with 1-acyl-2-[1-¹⁴C]arachidonoyl PC released¹⁴C-label into the medium upon further incubation. This release was slightly stimulated by zymosan and threefold higher in the presence of the Ca²⁺-ionophore A₂₃₁₈₇. Labeling of macrophages with intact phospholipid molecules appears to be a suitable method for studying acyl chain redistribution and release reactions.     

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.     

Modulation of fatty acid incorporation and desaturation by trifluoperazine in fungi

The effects of trifluoperazine (TFP) on [1-¹⁴C]fatty acid incorporation into the lipids ofMortierella ramanniana var.angulispora were studied. TFP decreased [1-¹⁴C]-fatty acid incorporation into phosphatidylcholine, phosphatidylethanolamine and triacylglycerol, but greatly increased¹⁴C-labeling in phosphatidic acid. These changes in [1-¹⁴C]fatty acid incorporation induced by TFP were accompanied by a decrease in desaturation of some [1-¹⁴C]fatty acids taken up by the fungal cells. When [1-¹⁴C]lioleic acid (LA) was incubated with the fungal cells, total γ-linolenic acid (GLA) formation from incorporated [1-¹⁴C]LA decreased, but the¹⁴C-labeled GLA conent in individual lipid classes was essentially unchanged. This suggests that the site of the TFP effect on GLA formation from [1-¹⁴C]LA taken up from the medium is not the desaturase acting on LA linked to complex lipids. On the other hand, GLA formation from [1-¹⁴C]oleic acid was much less susceptile to TFP, which suggests that in this fungus Δ6 desaturation to GLA has at least two different pathways with different degrees of susceptibility to TFP.     

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.     

Pyryliumverbindungen. XXVII. Spezifisch deuterierte Carbo- und Heterocyclen via 2,4,6-Triaryl-[3,5-2H2]pyryliumsalze

Pyrylium Compounds. XXVIII. Specifically Deuterated Carbo- and heterocycles via 2,4,6-Triaryl[3,5-²H₂]pyrylium Salts On heating with catalytic amounts of bases(e.g. triethyl amine) in deuterated alcohols such as methan[²H]ol or ethan[²H]ol pseudobases of 2, 4, 6-triarylpyrylium salts 1 undergo fast¹H/²H isotopic exchange reaction affording 1, 3, 5-triaryl[2, 4, 4-²H₃]pent-2-ene-1,5-diones which with [²H] perchloric acid give highly deuterated 2, 4, 6-triaryl-[3, 5-²H] pyrylium perchlorates 8 . These salts are obtainable also directly from 1 through a one-pot procedure by ring opening of the latter with deuterium oxide under the above-mentioned¹H/²H isotopic exchange conditions followed by recyclization of the formed 1, 3, 5-triaryl[2, 4, 4-²H₃]pent-2-ene-1, 5-diones with [²H]ClO₄. Ring transformations of 2, 4, 6-triphenyl[3, 5-²H²]pyrylium perchlorate (8a) to 2, 4, 6-triphenyl[3, 5-²H₂]nitrobenzene (9) 2, 4, 6-triphenyl[3, 5-²H₂]pyridine (10) , 1, 2, 4, 6-tetraphenyl[3, 5-²H₂]pyrydinium perchlorate (11) , 2, 4, 6-triphencyl[3, 5-²H₂]thiopyrylium perchlorate (12) , 2-benzoyl-3, 5-diphenyl[4-²H]furan (13) , and 3, 5, 7-triphenyl-[4, 4, 6-²H₃]4H-1, 2-diazepin (14) demonstrate the usability of pyrylium salts of type 8 as starting materials for syntheses of specifically deuterated carbo- and hetrocycles.     

Lipid composition and erucic acid in rat liver cells in culture

Erucic acid (Δ¹³-docosenoic acid) was added to fetal calf serum, then fed to rat liver epithelial cells in culture, and uptake measured at intervals over 24 hr. During the first 6 hr. of incubation, uptake of the docosenoic acid was 21 nmoles/hr/mg protein in 7-day cells, and 15 mmoles/hr/mg protein in 14-day cells. Of¹⁴C-labeled erucic acid taken up by the cells in 24 hr, radioactivity measurements showed 60% of the total lipid¹⁴C activity derived from [1-¹⁴C] 22∶1 in neutral lipid (NL) and 40% in phospholipid (PL); whereas 55% of lipid¹⁴C activity was in NL and 45% in PL when the substrate was [14-¹⁴C] 22∶1. Within the NL fraction, 75% of¹⁴C activity derived from [1-¹⁴C] 22∶1 was in triglyceride (TG) and 11% in cholesterol (CHL), while 79% was in TG and 6.5% in CHL when the substrate was [14-¹⁴C] 22∶1. Triglycerides and cholesteryl esters accumulated in the cells during incubation with erucic acid. Among phospholipids separated by thin layer chromatography, 75% of¹⁴C activity was in lecithin (PC), 10% in phosphatidylethanolamine (PE), 5% in sphingomyelin (SPH), and 1% or less in cardiolipin (DPG). The highest specific activity (SA) was in PC, followed by SPH and PE. Incubation with erucic acid altered fatty acid composition of PC, PE, and SPH, although amounts of phospholipids were unaffected. Gas liquid chromatography analyses detected 18% erucic acid in PC, 2% in PE, and 4–5% in SPH.     

Metabolism of linoleic acid in the cat

Cats fed a diet containing linoleate as the only polyunsaturated fatty acid showed extremely low levels of arachidonate in the plasma lipids, as well as an increase in linoleate, eicosadienoate and an unknown fatty acid. Administration of [1-¹⁴C] linoleic acid and [2-¹⁴C] eicosa-8,11,14-trienoic acid to cats showed that in the liver there was no conversion of the [1-¹⁴C] 18∶2 to arachidonate, whereas there was significant metabolism of [2-¹⁴C] 20∶3 to arachidonate. It was found when methyl-γ-linolenate was fed to cats that the level of 20∶3ω6 and 20∶4ω6 in the erythrocytes increased significantly. These results show that there is no significant Δ6 desaturase activity in the cat, whereas chain elongation and Δ5 desaturase enzymes are operative. The unknown fatty acid was isolated from the liver lipids and shown to be a 20-carbon fatty acid with 3 double bonds and which by gas liquid chromatography could be separated from 20∶3ω9 and 20∶3ω6. The presence of the Δ5-desaturase activity and the results of the ozonolysis studies indicated that this unknown fatty acid was eicosa-5,11,14-trienoic acid.