Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1-¹⁴C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1-¹⁴C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether–ethyl acetate–ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow (R_f) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1-¹⁴C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.     

Prostaglandin E2, prostaglandin E1 and thromboxane B2 release from human monocytes treated with C3b or bacterial lipopolysaccharide

C3b or lipopolysaccharide treatment of human peripheral blood monocytes in culture stimulates an early release of thromboxane B₂ and a delayed release of prostaglandin E into culture supernatants. Immunoreactive thromboxane B₂ release is maximal from 2–8 h, whereas prostaglandin E release is maximal from 16–24 h after stimulation of monocytes in culture. We further examined this process by comparing the time course of labelled prostaglandin E₂, prostaglandin E₁ and thromboxane B₂ release from human monocytes which were pulse or continuously labelled with [³H]arachidonic acid and [¹⁴C]eicosatrienoic acid. The release of labelled eicosanoids was compared with the release of immunoreactive prostaglandin E and thromboxane B₂. The time course of prostaglandin E₂ release was virtually identical to the release of prostaglandin E₁ in all culture supernatants regardless of labelling conditions. However, release of immunoreactive prostaglandin E paralleled the release of labelled prostaglandin E₁ and E₂ only for continuously labelled cultures. The release of labelled prostaglandin E₁ and E₂ from pulse labelled cultures paralleled the release of thromboxane B₂ and not immunoreactive prostaglandin. In contrast, labelled and immunoreactive thromboxane B₂, quantitated in the same culture supernatants, demonstrated similar release patterns regardless of labelling conditions. These findings indicate that the differential pattern of prostaglandin E and thromboxane B₂ release from human monocytes is not related to a time-dependent shift in the release of prostaglandin E₁ relative to prostaglandin E₂. Because thromboxane B₂ and prostaglandin E₂ are produced through cyclooxygenase mediated conversion of arachidonic acid, these results further suggest that prostaglandin E₂ and thromboxane B₂ are independently metabolized in human monocyte populations.     

Prostaglandin F2α produced by rabbit renal slices is not a metabolite of prostaglandin E2

Slices of rabbit renal medulla and rabbit renal papilla were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15-³H]-arachidonic acid. In both tissues, comparison of the isotope ratios of the radioactive products with the isotope ratio of the added arachidonic indicated that: (a) there was no discernable isotope effect in the biosynthesis of prostaglandin E₂; (b) prostaglandin F₂α was formed by reduction of prostaglandin H₂ and not by reduction of prostaglandin E₂; and (c) most of the radioactive product arose from arachidonic acid that had been incorporated into the tissue and not from the direct action of cyclooxygenase on arachidonic acid in the medium.     

Enrichment of human platelet phospholipids with linoleic acid diminishes thromboxane release

We have investigated whether exposure of human platelets to elevated concentrations of linoleic acid, the principal dietary polyunsaturate, would influence platelet thromboxane A₂ release. Platelets were incubated with albumin-bound linoleic acid at 30°C for 24 h, with prostaglandin E₁ added to prevent aggregation. The linoleic acid supplemented platelets released, on averaged, 50% less thromboxane A₂ in response to stimulation with thrombin than corresponding control platelets. Other fatty acids were without appreciable effect. The inhibition of thrombin-stimulated thromboxane A₂ release was dependent on the time and temperature of incubation, as well as on the concentration of added linoleic acid. Supplementation increased the amount of linoleic acid in the platelet phospholipids, but the arachidonic acid content of the phospholipids was reduced. [1-¹⁴C]Linoleic acid was not converted to arachidonic acid by the platelets. Linoleic acid was released exclusively form the inositol phosphoglycerides when the enriched platelets were stimulated with thrombin. The linoleate-enriched platelets converted less [1-¹⁴C]arachidonic acid to all prostaglandin products, suggesting that the platelet cyclooxygenase was partially inhibited.     

Influence of short term dietary supplementation of different lipids on aggregation and arachidonic acid metabolism in rabbit platelets

Semisynthetic diets containing 8% by weight of either corn oil or butter were fed to male New Zealand rabbits for three weeks. The plasma cholesterol values were determined, the threshold concentrations for aggregation of platelet rich plasmas were measured for collagen and Na arachidonate, and the conversion of ¹⁴C arachidonic acid to thromboxane B₂ and hydroxy fatty acids (HETE and HHT) at 10, 20 and 40 μM substrate concentrations were studied. The thresholds for arachidonate induced aggregation were lower and the amplitudes of collagen induced aggregations were greater in the butter fed than in the corn oil fed rabbits. Conversions of arachidonic acid to thromboxane B₂ but not to hydroxy fatty acids were greater in the butter fed rabbits at 10 and 20 μM substrate. The observed changes were accompanied by only slight modifications of plasma cholesterol levels.     

Elongation of acyl-CoAs by microsomes from etiolated leek seedlings

Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2-¹⁴C]malonyl-CoA was incorporated into fatty acids of C₁₆C₂₆. The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C₁₂C₂₄) elongation showed that stearoyl-CoA, in the presence of [2-¹⁴C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C₂₀C₂₆. [1-¹⁴C]C₁₆CoA and [1-¹⁴C]C₁₈CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1-¹⁴C]C₁₈CoA was a better primer than [1-¹⁴C]C₁₆CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1-¹⁴C]C₁₈CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2-¹⁴C]malonyl-CoA in the presence of C₁₈CoA, on the one hand, and [1-¹⁴C]C₁₈CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA.     

Transformation of arachidonic acid in the rat anterior pituitary

Rat anterior pituitaries were incubated with [1-¹⁴C]-arachidonic acid. The metabolites were purified by reversed-phase high pressure liquid chromatography. Conclusive identification of the compounds was performed by gas chromatography-mass spectrometry. The major metabolite of arachidonic acid was the 12-hydroxy-5,8,10,14-icosatetraenoic acid (0.1% of added radioactivity). Smaller amounts of 12-hydroxy-5,8,10-heptadecatrienoic acid and of 15-hydroxy-5,8,11,13-icosatetraenoic acid (0.01% of added radio-activity) were also isolated. Trace amounts of prostaglandins E₂, D₂ and F₂α were detected.     

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B₂ both from [1-¹⁴C]arachidonic acid added to washed platelets and from [1-¹⁴C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B₂ from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B₂ in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B₂ (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B₂ and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B₂ formation by the elastin.     

Dietary n-3 fatty acids,curcumin and capsaicin lower the release of lysosomal enzymes and eicosanoids in rat peritoneal macrophages

Male Wistar rats (12 rats/group) were fed a diet containing 8 wt % coconut oil or groundnut oil or cod-liver oil for a total period of 8 weeks. The diets were also supplemented with 2 wt % groundnut oil for providing essential fatty acids. During the last 2 weeks, 6 rats form each group were additionally given curcumin (30 mg/kg body wt/day) or capsaicin (5 mg/kg body wt/day) in 1 ml groundnut oil. The peritoneal macrophages from rats fed cod-liver oil diet secreted lower levels of lysosomal enzymes collagenase, elastase and hyaluronidase as compared to those from rats fed coconut oil or groundnut oil diets. Curcumin and capsaicin significantly lowered the secretion of these lysosomal enzymes from macrophages in animals given coconut oil or groundnut oil diet. Macrophages from rats fed cod-liver oil secreted lower amounts of prostaglandin E₂, 6-keto PGF_1a, leukotrienes B₄and C₄and also incorporated lesser amounts of [^3H]-arachidonic acid as compared to those given coconut oil or groundnut oil diets. Curcumin and capsaicin lowered the secretion of these eicosanoids and decreased the incorporation of [³H]-arachidonic acid in macrophage lipids. However curcumin and capsaicin significantly increased the secretion of 6-keto PGF_1ain all the groups of animals. These studies indicated that dietary cod-liver oil (rich in n-3 fatty acids), and spice principles curcumin and capsaicin can lower the secretory functions of macrophages in a beneficial manner.     

Metabolism of Arachidonic Acid to Epoxyeicosatrienoic Acids. Hydroxyeicosatetraenoic Acids, and Prostaglandins in Cultured Rat Hippocampal Astrocytes

Abstract: We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxy-genase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [³H]-arachidonic acid, formed products that eluted in four major groups designated as R_17–30, R_42–50, R_51–82, and R_83–90 based on their retention times in reverse-phase HPLC. These fractions were further segregated into as many as 13 peaks by normal-phase HPLC and a second reverse-phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact-mass spectrometry. Based on HPLC retention times and gas chromatography/electron impactmass spectrometry analysis, the more polar fractions (R_17–30) contained prostaglandin D₂ as the major cyclooxygenase product. Minor products included 6-keto prostaglandin F_1α, prostaglandin E₂, prostaglandin F_2α, and thromboxane B₂. Fractions R_42–50, R_51–82. and R_83–90 contained epoxygenase and lipoxygenase-like products. The major metabolite in fractions R_83–90 was 5, 6-epoxyeicosatrienoic acid (EET). Fractions R_51–82 contained 14, 15-and 8, 9-EETs, 12-and 5-hydroxyeicosatetraenoic acids, and 8, 9-and 5, 6-dihydroxyeicosatrienoic acids (DHETs). In fractions R_42–50, 14, 15-DHET was the major product. When radiolabeled [³H]14, 15-EET was incubated with astrocyte homogenate, it was rapidly metabolized to [³H]14, 15-DHET. The metabolism was inhibited by submicromolar concentration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxyalcohols from 14, 15-DHET was not observed. In conclusion, astro-cytes readily metabolize arachidonic acid to 14, 15-EET, 5, 6-EET, and their vicinal-diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.     

Apparent specificity of the thrombin-stimulated deacylation of endothelial glycerolipids for polyunsaturated fatty acids with a delta-5 desaturation

Human umbilical vein endothelial cells readily incorporate exogenous polyunsaturated fatty acids. Subsequent stimulation with thrombin results in the release of both arachidonate and eicosapentaenoate from cellular phospholipids. The present study has investigated the utilization of 8,11,14-[¹⁴C]eicosatrienoate, the precursor of prostaglandin E₁. Analysis of released ¹⁴C-fatty acids by radio-gas chromatography indicated that thrombin stimulated the release of 6–10% of the [¹⁴C]arachidonate synthesized by desaturation of the [¹⁴C]eicosatrienoate, but did not stimulate release of [¹⁴C]eicosatrienoate per se (less than 1%). As determined by digestion of cellular lipid extracts with pancreatic phospholipase A₂, both 8,11,14-[¹⁴C]eicosatrienoate and [¹⁴C]arachidonate were esterified primarily in the 2-position. Similarly, separation of phospholipid classes by two-dimensional thin-layer chromatography did not indicate any major differences in the distribution of the incorporated ¹⁴C-fatty acids. Experiments with additional ¹⁴C-fatty acids indicated that 5,8,11-eicosatrienoate is released in response to thrombin but that 8,11,14,17-eicosatetraenoate is not. These results suggest that the delta-5 double bond is required for the thrombin-stimulated release of free fatty acids from endothelial phospholipids and their subsequent availability as substrates for eicosanoid synthesis.     

Comparison of acetate- and pyruvate-dependent fatty-acid synthesis by spinach chloroplasts

In recent studies using intact chloroplasts of spinach (Spinacia oleracea L.) to investigate the accumulation of acetyl-CoA produced by the activity of either acetyl-CoA synthetase (EC 6.2.1.1) or the pyruvate-dehydrogenase complex, this product was not detectable. These results in combination with new information on the physiological levels of acetate and pyruvate in spinach chloroplasts (H.-J. Treede et al. 1986, Z. Naturforsch. 41 C, 733–740) prompted a reinvestigation of the incorporation of [1-¹⁴C] acetate and [2-¹⁴C] pyruvate into fatty acids at physiological concentrations.The K _m for the incorporation into fatty acids was about 0.1 mM for both metabolites and thus agreed with the values obtained by H.-J. Treede et al. (1986) for acetyl-CoA synthetase and the pyruvate dehydrogenase complex. However, acetate was incorporated with a threefold higher V _max. Saturation for pyruvate incorporation into the fattyacid fraction was achieved only at physiological pyruvate concentrations (<1.0 mM). The diffusion kinetics observed at higher concentrations may be the result of contamination with derivates of the labeled substrate. Competition as well as double-labeling experiments with [³H]acetate and [2-¹⁴C]pyruvate support the notion that, at least in spinach, chloroplastic acetate is the preferred substrate for fatty-acid synthesis when both substrates are supplied concurrently (P.G. Roughan et al., 1979 b, Biochem. J. 184, 565–569).Experiments with spinach leaf discs confirmed the predominance of fatty-acid incorporation from acetate. Radioactivity from [1-¹⁴C]acetate appeared to accumulate in glycerolipids while that from [2-¹⁴C]pyruvate was apparently shifted in favor of the products of prenyl metabolism.Abbreviations Chl chlorophyll - TLC thin-layer chromatography     

Relationship between the pentose-phosphate pathway and the de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cultures

This study focuses on the activity of the pentose-phosphate pathway and its relationship to de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cell cultures derived from 1-week old rat brain. The proportion of glucose that was metabolized along the pentose-phosphate pathway was estimated by measuring ¹⁴CO₂ production from [1-¹⁴C]-, [2-¹⁴C]- and [6-¹⁴C]glucose, the utilization of glucose and the production of lactate. Incorporation of ¹⁴C from [¹⁴C]glucose and from [3-¹⁴C]acetoacetate into lipids was analysed. The pentose- phosphate pathway produced much more CO₂ from glucose than the Krebs cycle, although it accounted for only a small part of the consumption of glucose (< 3%). The higher ¹⁴CO₂ production from [2-¹⁴C]glucose than from [6-¹⁴C]glucose indicated that recycling of the products of the pentose-phosphate pathway takes place in these cells.Gradual inhibition of the pathway with increasing concentrations of 6-aminonicotinamide resulted in a parallel inhibition of the conversion of acetoacetate and of glucose into fatty acids and into cholesterol. Glycolysis was also strongly inhibited in the presence of 6-aminonicotinamide whereas the activity of the Krebs cycle was not affected.These results suggest that de novo synthesis of fatty acids and cholesterol by oligodendrocytes of neonatal rats is closely geared to the activity of the pentose-phosphate pathway in these cells.     

Isolation of a new lipoxygenase metabolite of arachidonic acid, 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid from human platelets

Washed human platelets incubated with 1-¹⁴C -arachidonic acid (1mM) produced a new metabolite which migrated on thin layer chromatography close to thromboxane B₂, but which was identified by mass spectrometry as a trihydroxy fatty acid. The mass spectrum was consistent with the assigned structure, 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid (THETE). Platelet THETE synthesis from arachidonate was not inhibited by preincubation with aspirin or indomethacin but was blocked by 5,8,11,14-eicosatetraynoic acid. Therefore, THETE appears to arise via the platelet lipoxygenase pathway rather than via the prostaglandin cyclooxygenase. Two proposed structures, including a novel dihydro-hydroxy-pyran cyclic intermediate, which could give rise to THETE are presented.     

Fatty Acid Transport Through the Blood-Brain Barrier

Across the cerebral capillaries, the anatomical locus of the blood-brain barrier, the unidirectional influxes of the saturated fatty acids, octanoic and myristic acids, and the unsaturated essential fatty acid, linoleic acid, were measured. Employing an in situ rat brain perfusion technique that allows control of perfusate composition and accurate measurement of perfusate-to-brain fatty acid transport, we found that both [¹⁴C]octanoic and [¹⁴C]myristic acids were transported through the blood-brain barrier in vivo, in large part, by a specific, probenecid-sensitive transport system. However, the transport of [¹⁴C]linoleic acid was not probenecid sensitive. With 0.5 μM fatty acid but no plasma proteins in the perfusate, the permeability-surface area constant was higher for myristic acid (4.8 × 10^--²× s^-1) than for octanoic and linoleic acids (1.5 and 1.2 × 10^--²× s^-1, respectively). Approximately 70, 30, and 25% of the [¹⁴C]myristic, [¹⁴C]octanoic, or [¹⁴C]linoleic acids, respectively, were extracted from the perfusate.     

Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain

The metabolism of oligodendrocytes has been studied using cultures of oligodendrocyte-enriched glial cells isolated from cerebra of 5–8-day old rats. Cultures containing 60–80% oligodendrocytes were incubated for 16h with [3-¹⁴C]acetoacetate, d-[3-¹⁴C]3-hydroxybutyrate, [U-¹⁴C]glucose, l-[U-¹⁴C]glutamine and [1-¹⁴C]pyruvate or [2-¹⁴C]pyruvate in the presence or absence of other oxidizable substrates. Labelled CO₂ was collected as an index of oxidative metabolism and the incorporation of label into total lipids, fatty acids and cholesterol was used as an index of the de novo synthesis of lipids. Glucose, acetoacetate, D-3-hydroxybutyrate, pyruvate and l-lactate were measured to determine substrate utilization and product formation under various conditions. Our results indicate that glucose is rapidly converted to lactate and is a relatively poor substrate for oxidative metabolism and lipid synthesis. Ketone bodies were used as an energy source and as precursors for the synthesis of fatty acids and cholesterol. Preferential incorporation of acetoacetate into cholesterol was not observed. Exogenous pyruvate was incorporated into both the glycerol skeleton of complex lipids and into cholesterol and fatty acids. l-Glutamine appeared to be an important substrate for the energy metabolism of these cells.     

The Metabolism of the Germinating Oil Palm (Elaeis guineensis) Seedling : In Vivo Studies

The metabolism of ¹⁴C-labeled fatty acids and triacylglycerols was followed in intact germinating oil palm seedlings as well as in tissue slices. In the germinating seedling, the shoot contained a normal pattern of membrane fatty acids (mainly C₁₆, C_18:1, C_18:2) but the kernel contained about 68% C₁₂ and C₁₄ fatty acids. Haustorium fatty acids were intermediate between the two. [¹⁴C]Acetate was actively metabolized by shoot and haustorium slices but not so actively by the kernel. Approximately 9% to 17% was converted to water-soluble substances, 4% to 6% to CO₂, and 0.5% to 5.9% to lipids. The fatty acids synthesized in the shoot and haustorium were mainly C₁₆, C₁₈, and C_18:1 fatty acids but in the kernel about 18% to 32% of the ¹⁴C-fatty acids were C₁₂ fatty acids.

[¹⁴C]Lauric acid was absorbed and metabolized by haustorium slices and by the haustorium in intact seedlings; it was partly esterified to triacylglycerols and also converted to water-soluble substances and insoluble tissue material. In contrast, tri-[¹⁴C]laurin was absorbed but not metabolized. The haustorium also absorbed other fatty acids but the longer chain (C₁₆ and C₁₈) fatty acids were not esterified or metabolized further. Preincubation of the haustorium with plant hormones or in the presence of kernel tissue did not alter its inactivity towards tri-[¹⁴C]laurin.

When tri-[¹⁴C]laurin or [¹⁴C]lauric acid were injected into the seed or the shoot, there was no movement or radioactivity to other parts of the seedling. When injected into the shoot, but not into the seed, tri-[¹⁴C] laurin was hydrolyzed and partly metabolized to water-soluble substances.

     

Intermediary Metabolism of Organic Matter in the Sediments of a Eutrophic Lake

The rates, products, and controls of the metabolism of fermentation intermediates in the sediments of a eutrophic lake were examined. ¹⁴C-fatty acids were directly injected into sediment subcores for turnover rate measurements. The highest rates of acetate turnover were in surface sediments (0- to 2-cm depth). Methane was the dominant product of acetate metabolism at all depths. Simultaneous measurements of acetate, propionate, and lactate turnover in surface sediments gave turnover rates of 159, 20, and 3 μM/h, respectively. [2-¹⁴C]propionate and [U-¹⁴C]lactate were metabolized to [¹⁴C]acetate, ¹⁴CO₂, and ¹⁴CH₄. [¹⁴C]formate was completely converted to ¹⁴CO₂ in less than 1 min. Inhibition of methanogenesis with chloroform resulted in an immediate accumulation of volatile fatty acids and hydrogen. Hydrogen inhibited the metabolism of C₃-C₅ volatile fatty acids. The rates of fatty acid production were estimated from the rates of fatty acid accumulation in the presence of chloroform or hydrogen. The mean molar rates of production were acetate, 82%; propionate, 13%; butyrates, 2%; and valerates, 3%. A working model for carbon and electron flow is presented which illustrates that fermentation and methanogenesis are the predominate steps in carbon flow and that there is a close interaction between fermentative bacteria, acetogenic hydrogen-producing bacteria, and methanogens.     

Uptake,incorporation and calcium-ionophore-stimulated mobilization of arachidonic,eicosapentaenoic and docosahexaenoic acids by leucocytes of the rainbow trout,Salmo gairdneri

Rainbow trout leucocytes contain high levels of neutral lipid (about 70% of total lipid on a wt% basis) consisting of mostly triacylglycerol, free sterols and sterol esters (25%, 15% and 52% of neutral lipid, respectively). The phospholipids, separated by thin-layer chromatography, consisted predominantly of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, each present at about 30% of the total phospholipid. Radiolabelling of the leucocytes for 1 h with 1 μCi (approx. 6 μM) [1−¹⁴C]20:4(n−6), [1−¹⁴C]20:5(n−3) or [1−¹⁴C]22:6(n−3) each gave similar uptake values (approx. 1 · 10⁵ cpm/10⁷ leucocytes). The incorporation into total phospholipids was highest for 22:6(n−3) and lowest for 20:4(n−6). A higher percentage of radiolabel from [1−¹⁴C]22:6(n − 3) was found incorporated into phosphatidylcholine and phosphatidylethanolamine as compared to that from [1−¹⁴C]20:4(n − 6) and [1−^14C]20:5(n−3), while the reverse situation was found with phosphatidylinositol and phosphatidylserine. The relative rates of incorporation into the different phospholipid classes for all three fatty acids were in the order phosphatidylinositol > sphingomyelin > diphosphatidylglycerol > phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine. Calcium ionophore-challenge did not significantly alter the pattern of phospholipid radiolabel. Ionophore-challenge released large amounts of radiolabel, much of which was recovered after high-performance liquid chromatographic separation as free fatty acid/monohydroxy fatty acids, although only approx. 0.3% was recovered in leukotriene B₄ and leukotriene B₅ for the [1−¹⁴C]20:4(n−6) and [1−¹⁴C]20:5(n−3) labelled leucocytes, respectively. Other lipoxygenase products were also radiolabelled and tentatively identified as 20-carboxy-LTB₄, 20-hydroxy-LTB₄, 6-trans-LTB₄, 6-trans-12-epi-LTB₄, 6-trans-8-cis-12-epi-LTB₄ and the corresponding LTB₅ structures. No ‘6-series’ leukotrienes were produced from [1−¹⁴C]22:6(n−3), nor was there any evidence for the synthesis of ‘5-series’ leukotrienes via retroconversion of 22:6(n−3) to 20:5(n−3). This latter finding shows that, despite the preponderance of 22:6(n−3) in the membranes of trout leucocytes, this fatty acid is not a substrate for leukotriene generation.