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Results for “FATTY ACIDS, RADIOACTIVE”

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PubMed1979

Specificity of phospholipases in methylcholanthrene-transformed mouse fibroblasts activated by bradykinin, thrombin, serum, and ionophore A23187.

Methylcholanthrene-transformed mouse fibroblasts synthesize prostaglandins in response to bradykinin, thrombin, serum, and the ionophore A23187. These agents activate phospholipases, thereby releasing fatty acids from phospholipids. To examine the phospholipid specificity of the phospholipases activated by bradykinin, thrombin, serum, and A23187, cells were labeled with [14C]arachidonic acid and stimulated with these agents in the presence of delipidated bovine serum albumin. Phospholipid classes were resolved by two-dimensional chromatography on silica gel-coated paper. Only phosphatidylinositol and phosphatidylcholine lost radioactivity upon stimulation. To characterize the fatty acid specificity of the phospholipases, cells were incubated with 14C-labeled stearic, oleic, linoleic, eicosatrienoic, or arachidonic acid and then exposed to the stimuli. Bradykinin, thrombin, and serum caused specific release of radioactivity into the medium only from cells labeled with arachidonic acid or eicosatrienoic acid, whereas A23187 caused release from cells labeled with any one of the five fatty acids. We conclude that bradykinin, thrombin, and serum activate phospholipases that specifically hydrolyze arachidonyl and eicosatrienoyl phosphatidylinositol and phosphatidylcholine, whereas A23187 is less specific activator of phospholipases.

Animals↗

PubMed1982

Effect of albumin, low temperature and metabolic inhibitors on transport of fatty acids into cultured human leukemic myeloid cells.

Radioactively-labelled palmitic acid was used to study the effects of albumin, low temperature and several inhibitors of metabolism on transport of fatty acids into cultured human leukemic myeloid cells. When serum or albumin were present in the medium, uptake of fatty acid by cells as well as its further incorporation into phospholipids and neutral lipids were considerably reduced. Uptake and metabolic utilization of this fatty acid was reduced at low temperature, in the presence or absence of albumin in the incubation medium. In absence of albumin, addition of iodoacetate, sodium cyanide or sodium azide had but little effect on the total uptake of fatty acids while metabolic utilization was reduced. When albumin was present, these inhibitors reduced both total uptake and incorporation into lipids. The data suggest that incorporation of the fatty acid into the outer layer of the cell membrane is controlled by the concentration of free, uncomplexed molecules of fatty acid adjacent to the cell surface. In the absence of albumin this is a fast reaction which reaches nearly maximal uptake in three minutes. In the presence of albumin, this process is much slower and follows a nearly linear course between 3 and 60 minutes. Translocation into the inner layer of the membrane and subsequent utilization for metabolic processes is a much slower process, which seems to depend on the quantity of the fatty acid in the outer layer.

Azides↗

PubMedDate not supplied

[The effect of chronic alcoholic intoxication on the level and metabolism of glycolipids in the rat brain].

The chronic alcohol intoxication has been studied for its effect on the content of glycolipids in the rat brain and incorporation of [I-14C]acetate into them. It is established that administration of ethanol to animals (2 g per 1 kg of body weight daily for 7 days) rises the content of cerebrosides I in the brain tissue. The specific radioactivity of sulphatides I falls as a result of a decrease of the [I-14C]acetate into fatty acids and galactose. The specific radioactivity of sulphatides II, cerebrosides II and III falls as a result mainly of a decrease of the specific radioactivity in the galactose components.

Alcoholism↗

PubMed1985

Synthesis of C20 and C22 polyunsaturated fatty acids by the placenta of the sheep.

Incubation in vitro of sheep placental homogenates with [1-14C] oleic, linoleic and linolenic acids resulted in extensive amounts of radioactivity becoming associated with C20 and C22 polyunsaturated fatty acid products. The fatty acids of the fetal plasma displayed substantial levels of both C20 and C22 polyunsaturated fatty acids, including the n-9 eicosatrienoic fatty acid associated with essential fatty acid deficiency, but extremely low levels of linoleic and linolenic acids. A role for the placenta in the provision to the fetus of a range of C20 and C22 polyunsaturated fatty acids derived from C18 fatty acid precursors is implicated.

Animals↗

PubMed1975

Omega-cyclohexyl fatty acids in acidophilic thermophilic bacteria. Studies on their presence, structure, and biosynthesis using precursors labeled with stable isotopes and radioisotopes.

Omega-Cyclohexyl undecanoic acid and omega-cyclohexyl tridecanoic acid were found in 10 strains of acido-thermophilic bacteria isolated from different Japanese hot springs. These unusual fatty acids were found in the esterified form in glyceride type complex lipids and constituted 74 to 93% of the total fatty acids in the bacteria. The fatty acids other than omega-cyclohexyl fatty acids found were 14-methyl hexadecanoic acid (3 to 15%) and 15-methyl hexadecanoic acid (1 to 6%), and trace amounts of straight chain and methyl-branched tetra- and penta-decanoic acids. Biosynthesis of omega-cyclohexyl fatty acids increased with increase in the concentration of glucose in the culture medium. The metabolism of omega-cyclohexyl fatty acids was studied using deuterium-labeled precursors by mass fragmentation analysis. The deuterium of [2-D]glucose was specifically incorporated into position 2 of the cyclohexyl ring of the fatty acids, indicating that the ring was synthesized from the glucose molecule. Radioactivity was efficiently incorporated into the omega-cyclohexyl fatty acids from labeled glucose, shikimate, and cyclohexyl carboxylate. These findings indicate that omega-cyclohexyl fatty acids are synthesized with glucose through shikimic acid and probably cyclohexyl carboxylyl-CoA derivative as the intermediates.

Acetates↗

PubMed1991

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.

Animals↗

PubMed1986

The 73 kilodalton heat shock cognate protein purified from rat brain contains nonesterified palmitic and stearic acids.

A protein related to the 71 kilodalton inducible rat heat shock protein was purified to electrophoretic homogeneity in milligram amounts from brain tissue of nonheat-stressed rats. The protein has been designated as a stress cognate protein based on previous studies and data presented herein that this protein cross-reacted with a monoclonal antibody originally raised against the Drosophila 70 kilodalton heat shock protein. The purified protein had an apparent molecular mass of 73 kilodaltons when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent mass of 150 kilodaltons as determined by nondissociative gel chromatography, suggesting that the purified protein is a homodimer. The purified protein had isoelectric points of 5.0 under nondissociative conditions and 5.6 when exposed to protein denaturants, suggesting loss of bound anionic molecules and/or net exposure of basic residues upon denaturation. Chloroform/methanol extraction of the purified protein and subsequent analyses by thin layer and gas-liquid chromatography resulted in the identification of palmitic and stearic acids noncovalently bound to the protein. Approximately four molecules of fatty acids were bound per dimer with palmitic and stearic acids present in a one-to-one ratio. The purified protein did not bind exogenously added radioactive palmitate, indicating that the fatty acid-binding sites of the cognate protein were fully occupied and that the associated fatty acids were too tightly bound to exchange readily. The possible significance of the fatty acids associated with the 73 kilodalton stress cognate protein is discussed.

Animals↗

PubMed1992

Mobilization of diacylglycerol in intact HeLa cells by exogenous phospholipase C from Cl. perfringens is accompanied by release of fatty acids including arachidonic acid.

The second messenger diacylglycerol (DAG), chiefly derived from phosphatidylcholine (PC) or from phosphatidylinositol (PI), through the activation of specific phospholipases C (PLC), plays a key role in cellular stimulation. The activation of a particular PLC was simulated in intact HeLa cells by treatment with exogenous PC-PLC (Cl. perfringens) or with PI-PLC (B. cereus). Both enzymes rapidly mobilized DAG. However, only PC-PLC led, in Hela cells, to morphological changes (which were reversible on enzyme removal within the time frame of the experiments) and to an increase of intracellular calcium concentration with a lag of > 10 min. In cells prelabeled with [1-14C]arachidonic acid only PC-PLC but not PI-PLC induced the release of labeled fatty acid with a lag of > 10 min. Upon prelabeling of cells with [1-14C]oleic acid, PC-PLC led to a release of radioactive oleic acid. The release of arachidonic acid (AA) required a threshold dose of PC-PLC and a minimum time of treatment beyond which the AA release continued for a certain period, even in the absence of the exogenous enzyme. Under the conditions used, neither PLA2 nor DAG lipase activity were detectable in the PC-PLC preparation. Therefore, AA release was due to activation of a cellular enzyme, probably cellular PLA2 activity. The PC-PLC-induced AA release could be inhibited to a certain extent by EGTA and by quinacrine but not by the glucocorticoid fluocinolone acetonide. Only PC-PLC (but not PI-PLC) caused, in addition, an increase of the level of monoglycerol, which paralleled the appearance of AA. An increase of labeled monoglycerol was detectable in HeLa cells prelabeled with radioactive oleic acid or with 1-[1-14C]palmitoyl-lyso-PC but not in cells prelabeled with radioactive AA, thus indicating that the fatty acid originated from sn-2 position of the glycerol moiety. The 1-monoacylglycerol was probably generated from lysophospholipids by the bacterial PC-PLC. This enzyme preparation has been shown to catalyze such breakdown of lysophosphatidylcholine in vitro. PC-PLC-induced AA release occurred also after down-regulation of protein kinase C by an overnight pretreatment with phorbol ester TPA (TPA-pretreated cells, but not control cells, on treatment with PC-PLC, metabolized AA to prostaglandins).(ABSTRACT TRUNCATED AT 400 WORDS)

Arachidonic Acid↗

PubMed2000

A fatty acid-binding protein and a protein disulphide isomerase-related protein expressed in urochordate gonad cytosol.

Despite the evolutionary-tree data suggesting that gene duplication leading to the divergence of the three branches which heart, liver and intestinal fatty acid-binding proteins belong to must have occurred before the vertebrate/invertebrate split, only the heart fatty acid-binding protein has been reported for invertebrates. In an attempt to shed light on this apparent inconsistency the presence of the other two branch members was investigated in the Urochordata Molgula pedunculata, an ascidian species close to vertebrates. The mantle-, gonad- and digestive tube-cytosolic fractions, obtained by centrifugation at 106,000 g, were incubated separately with [1-(14)C]palmitic acid and then fractionated on a Sephadex G-75 column. In the case of gonads and digestive tube, radioactive peaks corresponding to a molecular mass of 14-16 kDa, characteristic of fatty acid-binding proteins, were detected. When the experiment was performed on the mantle, this peak showing fatty acid binding capacity was absent. Western Blot of the radioactive 14-16 kDa Sephadex fraction from the urochordate gonad cross-reacted with rat liver fatty acid-binding protein anti-serum but did not do so with anti-rat intestinal, adipocyte or heart fatty acid-binding protein antisera. The material from the digestive tube was not recognized by any of the antisera. The most abundant protein in said 14-16 kDa fraction was a protein disulphide isomerase-related protein. Its partial amino acid sequence was determined.

Amino Acid Sequence↗

PubMed1999

Radioactive fingerprinting of microorganisms that oxidize atmospheric methane in different soils.

Microorganisms that oxidize atmospheric methane in soils were characterized by radioactive labelling with (14)CH(4) followed by analysis of radiolabelled phospholipid ester-linked fatty acids ((14)C-PLFAs). The radioactive fingerprinting technique was used to compare active methanotrophs in soil samples from Greenland, Denmark, the United States, and Brazil. The (14)C-PLFA fingerprints indicated that closely related methanotrophic bacteria were responsible for the oxidation of atmospheric methane in the soils. Significant amounts of labelled PLFAs produced by the unknown soil methanotrophs coeluted with a group of fatty acids that included i17:0, a17:0, and 17:1omega8c (up to 9.0% of the total (14)C-PLFAs). These PLFAs are not known to be significant constituents of methanotrophic bacteria. The major PLFAs of the soil methanotrophs (73.5 to 89.0% of the total PLFAs) coeluted with 18:1 and 18:0 fatty acids (e.g., 18:1omega9, 18:1omega7, and 18:0). The (14)C-PLFAs fingerprints of the soil methanotrophs that oxidized atmospheric methane did not change after long-term methane enrichment at 170 ppm CH(4). The (14)C-PLFA fingerprints of the soil methanotrophs were different from the PLFA profiles of type I and type II methanotrophic bacteria described previously. Some similarity at the PLFA level was observed between the unknown soil methanotrophs and the PLFA phenotype of the type II methanotrophs. Methanotrophs in Arctic, temperate, and tropical regions assimilated between 20 and 54% of the atmospheric methane that was metabolized. The lowest relative assimilation (percent) was observed for methanotrophs in agricultural soil, whereas the highest assimilation was observed for methanotrophs in rain forest soil. The results suggest that methanotrophs with relatively high carbon conversion efficiencies and very similar PLFA compositions dominate atmospheric methane metabolism in different soils. The characteristics of the methane metabolism and the (14)C-PLFA fingerprints excluded any significant role of autotrophic ammonia oxidizers in the metabolism of atmospheric methane.

Carbon Radioisotopes↗

PubMed1975

Effect of halofenate and clofibrate on lipid synthesis in rat adipocytes.

The free acids of the plasma lipid-lowering agents, halofenate and clofibrate inhibited the incorporation of radioactive glucose and pyruvate into fatty acids of isolated adipocytes prepared from rat epididymal fat pads. The concentration which inhibited fatty acid synthesis was dependent on the bovine serum albumin concentration in the incubation. The 50 per cent inhibitory concentration of the free acid of halofenate in 1 per cent, 2 percent and 4 per cent albumin was 0.9 mM, 2.3 MM and 4.4 mM, respectively. The potency of clofibrate was also lowered by increasing the albumin concentration. These compounds inhibited the uptake of both [14C]glucose and [14C]pyruvate to the same degree as the incorporation of these substrates into fatty acids. However, the drugs either had no effect on , or stimulated the uptake of palmitate by the cells. Leucine accumulation by the adipocytes was unaffected by halofenate (free acid) and inhibited by clofibrate (free acid). A comparison of these agents with (minus)-hydroxycitrate, kynurenate and cerulenin (inhibitors of ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase, respectively) on the oxidation of pyruvate suggested that they inhibited pyruvate metabolism at or near the enzyme, pyruvate dehydrogenase.

Adipose Tissue↗

PubMed1983

The effect of concanavalin A on the incorporation of fatty acids into lymphocyte phospholipids.

The incorporation of radioactively labeled palmitic, stearic, oleic, linoleic and arachidonic acid into the phospholipids of lymphocytes was studied. When concanavalin A (Con A) was added a gradual increase in the incorporation was found over the period investigated (0-4 h). Changes were not detected over the first 15 min. The increased incorporation at 4 h was shown to be Con A dose dependent and the Con A concentrations for the maximum effect on fatty acid incorporation (at 4 h) and on [3H]thymidine incorporation (at 72 h) were the same. With the exception of stearic acid all the other fatty acids showed an increased incorporation into phosphatidyl choline on addition of Con A, linoleic and oleic acids also had an increased incorporation into phosphatidyl ethanolamine.

Animals↗

PubMed1978

Free radical damage produced by carbon tetrachloride in the lipids of various rat tissues.

Alterations produced by carbon tetrachloride in the lipids of extrahepatic tissues were studied. Consistent incorporation (about one-half of that occurring in the liver) of 14C from 14CCl4 was found in the phospholipids of the intestinal mucosa; kidney, adrenal and lung phospholipids were labeled to a lower extent, while spleen, testis, brain, heart and skeletal muscle lipids showed minor levels of radioactivity. The analysis of fatty acid methyl esters derived from phospholipids by gas-liquid chromatography (GLC) with an electron capture detector (ECD), showed ECD response similar to that observed with the liver, in the case of the intestinal mucosa and the kidney; on the contary no ECD response was seen with the organs, such as skeletal muscle, heart and testis in which the incorporation is almost negligible. Unlike liver phospholipids, the phospholipids of the intestinal mucosa and the kidney did not show diene conjugation absorption. Also, no evidence of lipid peroxidation was found in the thin-layer chromatographic analysis of fatty acid methyl esters prepared from the phospholipids of these organs. Possible meanings of these results are discussed.

Animals↗

PubMed1986

Comparative utilization of glycerol and alanine as liver gluconeogenic substrates in the fed late pregnant rat.

The appearance of plasma [14C]glucose in the inferior cava vein after a pulse of 0.2 mmol of [U-14C]L-alanine or [U-14C]glycerol/200 g body wt given through the portal vein was studied in fed 21 day pregnant rats and virgin controls under pentobarbital anesthesia. In both groups values were much higher when [U-14C]glycerol was the administered tracer than when [U-14C]L-alanine, and they were augmented in pregnant versus virgin animals at 1 min when receiving [U-14C]glycerol and at 2 min when receiving [U-14C]L-alanine. 20 min after the tracers rats receiving [U-14C]glycerol showed much higher liver [14C]glycogen and [14C]glyceride glycerol than those receiving [U-14C]L-alanine. Radioactivity present in liver as [14C]glyceride glycerol was greater in pregnant than in virgin rats receiving [U-14C]glycerol whereas radioactivity corresponding to [14C]fatty acids was lower in the former group receiving either tracer. At 20 min after maternal treatments fetuses showed lower plasma [14C]glycerol than [14C]alanine values but plasma [14C]glucose and liver [14C]glycogen values were much greater in fetuses from mothers receiving [U-14C]glycerol than [U-14C]L-amine. Besides showing the higher gluconeogenic efficiency in pregnant than in virgin rats, results indicate that at late gestation glycerol is used as a preferential substrate for both glucose and glyceride glycerol synthesis in liver.

Alanine↗

PubMed1975

Production of CO2 in the living mouse immediately after injection of 1- or 2-14C acetate.

Three groups of mice, normally fed, fasted and fed after a fasting period are injected intravenously with either 1- or 2-14C acetate. The respiratory 14CO2 as well as that of the liver, the adipose tissue and the carcass are collected after 3 min and the radioactivity measured. A determination is also made of the radioactivity of the tissue fatty acids and, for two groups of mice, of the circulating glucose. A calculation is suggested by which the number of revolutions performed by the acetate C in the Krebs cycle before it is transformed into CO2 can be deduced. The results suggest that the Krebs cycle is very open, that the acetate C found in the glucose has already broken away from the cycle after one revolution and that the C which appears in the form of CO2 has performed an average of only 2.8 to 3.6 revolutions. The results are evaluated as a function of the experimental conditions chosen.

Acetates↗

PubMed1971

Lipid metabolism by rat lung in vitro. Effect of starvation and re-feeding on utilization of (U- 14 C)glucose by lung slices.

1. The incorporation of [U-(14)C]glucose into several lipid components of lung and liver slices, and the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ;malic' enzyme (EC 1.1.1.40) and NADP-isocitrate dehydrogenase (EC 1.1.1.42) of the cell cytosol were examined in normal, starved and re-fed rats. 2. Lipogenesis and the activities of these enzymes in liver were decreased markedly in rats starved for 72h. Re-feeding starved rats on a fat-free diet for 72h resulted in the well documented hyperlipogenic response in liver, particularly in its ability to convert glucose into neutral lipid, and increased activities of glucose 6-phosphate dehydrogenase, ;malic' enzyme and 6-phosphogluconate dehydrogenase to values approx. 700, 470 and 250% of controls respectively. 3. Approx. 70% of the total label in lung lipids was present in the phospholipid fraction. Hydrolysis of lung phospholipids revealed that lipogenesis from glucose was considerable, with approx. 40% of the total phospholipid radioactivity present in the fatty acid fraction. 4. Incorporation of glucose into total lung lipids was decreased by approx. 40% in lung slices of starved rats and was returned to control values on re-feeding. Although phospholipid synthesis from glucose was decreased in lung slices of starved rats, the decrease proportionally was greater for the fatty acid fraction (approx. 50%) as compared with the glycerol fraction (approx. 25%). 5. The activities of lung glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-isocitrate dehydrogenase were not affected by the dietary alterations. ;Malic' enzyme activity was not detected in lung cytosol preparations. 6. The results are discussed in relation to the surface-active lining layer (surfactant) of the lung.

Animals↗

PubMed1987

Lipid metabolism in electroplax.

The in vivo labeling of electrocyte lipids is followed after injection of radioactive glycerol and two fatty acids, oleate and arachidonate, into the electric organ of an elasmobranch (Discopyge tschudii). De novo synthesis of lipids and acyl-exchange reactions are operative in the electrocyte. The three precursors are preferentially incorporated into phosphatidylcholine, phosphatidylinositol, and triacylglycerols. The highest specific activities are attained by triacylglycerols and polyphosphoinositides. Electrocyte stacks from electric organ show an efficient and continuous esterification of oleate and arachidonate into lipids after several hours of incubation. Except for an apparently more active labeling of triacylglycerols, which is attributed to the larger availability of free fatty acid precursors under the in vitro experimental conditions, the pattern of lipid labeling is similar to that attained in vivo. 32P-labeled lipids are also steadily produced in electrocyte stacks (24 h of incubation with [32P]phosphate) using glucose as the sole exogenous source of energy. Polyphosphoinositides are the lipids preferentially labeled. The ability to sustain the labeling of lipids under in vitro conditions renders isolated electrocyte stacks an interesting model for future research on lipid involvement in cholinergic function.

Animals↗

PubMed1984

Fat metabolism following an intravenous bolus dose of a fat emulsion and carnitine.

Intravenous fat tolerance tests were performed with (carboxyl-14C)-triolein labelled Intralipid in four normal subjects with and without L-carnitine administration, 20 and 25 mg/kg body weight. The pharmacokinetics of L-carnitine was studied simultaneously with measurements of variables reflecting fat metabolism during 4 h. 3-OH-butyrate concentration in plasma was higher in all subjects when carnitine was given. No effect of carnitine was found in elimination of the exogenous triglycerides, the 14CO2 activity in expired air, concentration and specific radioactivity of non- esterified fatty acids or glucose in plasma. The data suggest that carnitine may slightly increase fatty acid oxidation in normal subjects provided that increase of 3-OH-butyrate concentration in plasma is the most sensitive variable reflecting fatty acid oxidation of the variables applied in this study.

Adult↗