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Results for “FATTY ACIDS, RADIOACTIVE”

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PubMed1976

Effects of p-chlorophenoxyisobutyrate on myocardial free fatty acid extraction, ventricular blood flow, and epicardial ST-segment elevation during coronary occlusion in dogs.

The effect of p-chlorophenoxyisobutyrate (CPIB) on ST-segment elevation in epicardial electrocardiographic recordings was studied during coronary artery occlusion in dogs. Occlusion alone raised the sum of ST-segment elevations (sigmaST) to 26 +/- 6 mV (mean +/- SEM). Intravenous (i.v.) administration of CPIB 30 min before re-occlusion reduced sigmaST to 14 +/- 3 mV (P less than 0.03). A continuous i.v. infusion of isoproterenol increased sigmaST to 74 +/- 11 mV. Pretreatment with CPIB reduced sigmaST during isoproterenol infusion to 40 +/- 7 mV (P less than 0.005). CPIB had no effect on mean aortic blood pressure, heart rate, or regional myocardial blood flow, as measured by radioactive microspheres. Arterial free fatty acid (FFA) concentrations were reduced by CPIB from 466 +/- 41 to 221 +/- 44 muEq/L (P less than 0.001) in the basal state, and from 1966 +/- 183 to 1429 +/- 209 muEq/L (P less than 0.001) during isoproterenol infusion. The reduction in arterial FFA concentration was associated with a proportionate decrease in the myocardial extraction of FFA. Similar changes were observed when CPIB was administered during an occlusion which had been established 10 min earlier. These observations support other evidence that the severity of acute myocardial ischemic injury in dogs is positively correlated with the myocardial extraction of FFA, and that the severity of the ischemic injury can be reduced by effective antilipolytic therapy.

Animals↗

PubMed1995

Techniques for the measurement of white adipose tissue metabolism: a practical guide.

A number of old and new techniques to study various aspects of white adipose tissue metabolism in vivo and in vitro are discussed. It is possible to determine lipolysis rates in vivo with tracer techniques using glycerol or fatty acids labelled with stable or radioactive isotopes. These methods allow the determination of whole body lipolysis rates but are not valuable for the investigations of regional variations in lipolysis. When combined they permit a calculation of the rate of re-esterification of free fatty acids. The vein draining abdominal subcutaneous adipose tissue can be cannulated in humans. By this method substrate turnover can be determined in vivo over the cannulated adipose region. With microdialysis it is possible to study local metabolism in vivo in different adipose tissue regions. At the same time it is possible to locally manipulate the tissue with metabolically active pharmacological substances. A number of in vitro methods to determine glucose transport in isolated fat cells are developed. The most accurate one uses 3-O-methyl glucose as tracer. These methods can be combined with studies of the further metabolism of glucose to lipids, lactate and carbon dioxide using simple (usually radioactive) methods. Lipolysis as well as release and re-esterification of free fatty acids can be investigated in detailed in vitro with sensitive techniques based on luminescence. Finally, triglyceride turnover and partial metabolism of acylglycerols can be investigated in vitro with a double isotope technique.

Adipose Tissue↗

PubMed1997

Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase.

Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10-20 microM, assuming a stromal volume of 66 microl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 microM. Malonyl-CoA concentrations were also very low (<0.1-3.0 microM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 microM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA-acyl-carrier protein transacylase reactions at the concentrations of metabolites measured here are < 0.1-4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.

Acetyl Coenzyme A↗

PubMed1998

Astrocytes are mainly responsible for the polyunsaturated fatty acid enrichment in blood-brain barrier endothelial cells in vitro.

To determine the respective roles of endothelial cells from brain capillaries and astrocytes in the conversion of circulating 18:2n-6 and 18:3n-3 into 20:4n-6 and 22:6n-3, respectively, a coculture of the two cell types mimicking the in vivo blood-brain barrier was used. During the culture period, endothelial cells cultured on an insert were set above the medium of a Petri dish containing or not a stabilized culture of astrocytes. Five days after confluence, labeled 18:2n-6 and 18:3n-3 (10 microM each) were added to the endothelial cells and incubated for 48 h. Analogous experiments were also performed by using each cell type cultured alone in the culture device. The distribution of radioactivity in lipids and fatty acids was studied in all the compartments of the culture device. Endothelial cells cultured alone weakly converted the precursor fatty acids into 20:4n-6 and 22:6n-3. When endothelial cells were cocultured with astrocytes, their content of polyunsaturated fatty acids increased dramatically. This effect was associated with the uptake of polyunsaturated fatty acids from the lower medium (astrocyte medium). These fatty acids were released by astrocytes after they were synthesized from the precursor fatty acids that passed through the endothelial cell monolayer into the lower medium. Polyunsaturated fatty acids were released by astrocytes as unesterified fatty acids and as phospholipids (mainly phosphatidylcholine and lysophosphatidylcholine) even when the medium was devoid of serum. These results suggest that astrocytes could play a major role in the delivery of essential polyunsaturated fatty acids to the barrier itself and to the brain.

Animals↗

PubMed1999

Differential absorption and esterification of dietary long-chain fatty acids by larvae of the dragonfly, Aeshna cyanea.

In order to evaluate whether dietary long-chain fatty acids were differentially absorbed, Aeshna cyanea larvae received 5 microliters oral doses containing combinations of two radiolabeled fatty acids at nearly equal radioactive and nmolar concentrations: (1) 3H-oleic and 14C-palmitic acids; (2) 3H-oleic and 14C-stearic acids; and (3) 3H-palmitic and 14C-stearic acids. After 3 h or 1 day, hemolymph samples, midgut tissue, midgut contents and fat body tissue were collected and assayed for labeled fatty acids. The 3H/14C ratios indicated that there was a preference for absorption of the monounsaturated oleic acid over both saturated palmitic and stearic acids and that the shorter palmitic acid was absorbed at a higher rate than the longer stearic acid. There were also differences in the 3H/14C ratios of the various lipid classes of the midgut wall, hemolymph, and fat body that reflected differential esterifications and transport of these fatty acids.

Absorption↗

PubMed1983

Biosynthesis of long-chain polyenoic acids from arachidonic acid in cultures of enriched spermatocytes and spermatids from mouse testis.

Primary spermatocytes (PS), round spermatids (RS) and condensing spermatids (CS) from mouse testes were enriched on Sta-Put 1 X g density gradients and cultured for 22 or 44 hr in the presence of [1-14C]arachidonate. Mass and radioactivity were measured by gas radiochromatography of constituent fatty acids of the various complex lipid classes fractionated by thin layer chromatography. Patterns and levels of incorporation were compared with those of whole testis, both in vitro and in vivo. The 20:4, 22:4, 22:5, 24:4 and 24:5 of the germinal cells contained levels of radioactivity in each lipid class which were consistent with an important role for the germinal cells in long-chain polyenoic acid (LCPA) metabolism. Cells which represented later stages of spermatogenesis (RS, CS) incorporated much higher percentages and absolute amounts of radioactivity into the fatty acids derived from 20:4 by elongation-desaturation pathways than did PS or whole testis in vitro. These differences were most pronounced in triacylglycerol of CS. Distributions of mass and radioactivity among lipid classes suggest synthesis of triacylglycerol by CS with a high degree of specificity for 22 or 24 carbon LCPA at the sn-3 position.

Animals↗

PubMed1979

Gas liquid chromatographic studies of [1-14C] palmitate uptake by the perfused guinea-pig heart.

In addition to the uptake of palmitate by the isolated perfused guinea-pig heart, there was a substantial release of longer chain fatty acids into the perfusate amounting to about 50% of the uptake of palmitate. Both the uptake of palmitate and the release of other fatty acids was diminished in the presence of 95% N2. The specific activity of myocardial palmitate was increased under 95% N2. No significant difference in the distribution of radioactivity between the different myocardial fatty acids occurred between 95% O2 and 95% N2.

Animals↗

PubMed1989

Effect of oxygen deprivation on metabolism of arachidonic acid by cultures of rat heart cells.

To investigate the mechanisms responsible for the impairment of phospholipid metabolism observed in ischemic cells, we have studied the effect of conditions simulating ischemia on the metabolism of arachidonic acid (AA) by muscle (M-) and nonmuscle (F-) cells isolated from newborn rat hearts and cultured separately. In muscle cells, oxygen deprivation induces a significant stimulation of the release of [14C]AA from prelabeled cells associated with a preferential redistribution of [14C]AA into cell triglycerides but not formation of radioactive prostaglandins. Moreover, the fatty acid content of phospholipids, as measured by capillary gas chromatography, appears markedly reduced in ischemic myocardial cells. This fact may be related to phospholipase stimulation during ischemia as suggested by the antagonistic effect of mepacrine or p-bromophenacyl bromide. In contrast, oxygen deprivation failed to induce any significant alteration of AA metabolism in fibroblast-like heart cells. Our results indicate that these cultures of newborn rat heart cells, which exhibit many of the features observed in intact organ during ischemia, may represent a useful experimental model to investigate the pharmacological control of the membrane phospholipid turnover.

Aerobiosis↗

PubMed1975

Turnover of plasma oleic acid measured by radio-gas chromatography.

Gas-liquid chromatography with radioactivity detection (Radio-GLC) was investigated as an analytical means of determining the fractional turnover rates of plasma free fatty acids. For this purpose normal dogs were infused with 1.838 muCi/min of [1-14C]oleic acid complexed with albumin and plasma samples were taken at 0 to 110 minutes. The plasma free fatty acids were isolated by a modified Dole extraction and the methyl esters, prepared by diazomethylation, were identified and quantitated by GLC and radio-GLC using radioactive methyl heptadecanoate as internal standard. The study demonstrates that physiologically feasible infusion rates and loads of radioactive acids can be found which permit accurate analyses of plasma free fatty acids by radio-GLC. During a 2-hour infusion no labeled acid other than oleic appeared in plasma indicating that the method could be used to study the turnover of a mixture of fatty acids simultaneously. These results also indicate that conventional methods of determination of radioactivity in purified extracts can be employed without concern for recycling of label among the fatty acids, at least over short periods of time. The radio-GLC technique described yields approximately 20% higher fractional turnover times for oleic acid than do standard methods.

Animals↗

PubMed1978

Microbial transformation of 14C-labeled 2,4,6-trinitrotoluene in an activated-sludge system.

The fate of 14C-labeled 2,4,6-trinitrotoluene (TNT) in an activated-sludge system was investigated. No [14C]TNT could be detected in the contents of an aerated reactor after 3 to 5 days of incubation. No significant 14CO2 was formed, and the radioactivity was about equally divided between the floc and the supernatant. The radioactive carbon present in the microflora was mainly associated with the lipid and protein components, but the characteristic constituents of these compounds (e.g., fatty acids and amino acids) were not radioactive. The major part of the 14C present in the lipid and protein fractions was found in precipitates that formed in both fractions. The solubility properties and infrared spectra of these precipitates suggested that they are macromolecular structures of the polyamide type formed by the reaction of TNT biotransformation products with lipids, fatty acids, and protein constituents of the microbial flora. This hypothesis is further supported by the correspondence of the infrared spectrum of the lipid precipitate with that of a model compound synthesized from TNT transformation products and lipid precursors. The resistance of these macromolecules to further biodegradation was paralleled by the reported resistance to microbial attack of polyamides containing similar linkages.

Bacteria↗

PubMed1984

Platelets during alimentary hyperlipaemia induced by cream and cod liver oil.

Healthy male subjects were given 100 g lipids as cream or cod liver oil. They were examined before and during alimentary hyperlipaemia. Cream increased platelet coagulant activity in plasma and reduced it in washed platelets. Both meals increased platelet sensitivity to thrombin and collagen. Cod liver oil reduced the uptake of arachidonic acid (AA) in platelets. Washed platelets prelabelled with [14C]-AA increased radioactivity 10-fold in free fatty acids (FFA) by exposure to thrombin [2 U (10(9) platelets)-1] for 20 s. This increase was augmented by cod liver oil. Phosphatidylethanolamine and phosphatidylcholine were the most significant donors of AA during thrombin stimulation. By exposure of prelabelled washed platelets to thrombin [0.3 U (10(9) platelets)-1] for 4 min the percentage distribution of [14C]-AA increased after cod liver oil in FFA plus the products of AA metabolism formed by the cyclooxygenase and lipoxygenase pathways. This study indicates that intake of a meal rich in lipids induces acute disturbances in platelets that may favour thrombosis. These effects were observed after intake of both saturated and polyunsaturated fatty acids.

Adult↗

PubMed1980

Biosynthesis of ergosterol in cell-free system of yeast.

We previously proposed the occurrence of multiple pathways in the ergosterol biosynthesis of yeast, based on the results of examination of 14C-incorporation into sterols from L-[methyl-14C]methionine which was given to the intact cells of yeast. This led us to investigate the validity of the pathways by experiments with the cell-free system. Highly active cell-free extracts could be prepared by disruption of yeast cells with a Vibrogen Cell Mill in the presence of 0.1 mM dithiothreitol. This preparation catalyzed 14C-incorporation from [14C]methionine into ergosterol with a high yield. This preparation was found to be favorable for elucidation of ergosterol synthesis, since only a small amount of radioactivity was incorporated into fatty acid ester form of sterols which were reported to be inactive as a substrate for sterol synthesis reaction. Time course experiment of 14C-incorporation from [14C]methionine into various sterols under aerobic conditions showed that ergosta-8,24(28)-dien-3 beta-ol was a precursor for ergosta-7,24(28)-dien-3 beta-ol and that radioactivities were converted through ergosta-5,7,24(28)-trien-3 beta-ol and ergosta-5,7,22,24(28)-tetraen-3 beta-ol into ergosterol with time. In contrast, similar experiments under anaerobic conditions showed that ergosta-7,24(28)-dien-3 beta-ol accumulated and very little conversion of radioactivity into ergosterol occurred. In addition, the results indicated that oxygen was required for the introduction of double bond into 22 position as well as into 5 position. The results obtained with the cell-free system supported the validity of the proposal of multiple pathways of ergosterol synthesis in the intact cells.

Anaerobiosis↗

PubMed1981

Change of galactolipids and metabolism of fatty acids in the organotypic culture of myelinating mouse brain.

The concentrations of cerebrosides and sulfatides, myelin-characteristic galactolipids, can be determined in organotypic culture of newborn mouse cerebellum by high-performance liquid chromatography. These galactolipids can be detected at 9-days in vitro explants and increased steadily until the explants were more tan 3 weeks old. The levels of nonhydroxycerebroside, hydroxycerebroside, nonhydroxysulfatide, hydroxysulfatide, and monogalactosyl diacylglycerol were 0.3, 0.6, 0.3, 0.2, and 0.3 nmol/mg protein at 9 days in vitro, respectively, and 1.0, 2.7, 0.9, 0.4, and 0.6 at 21 days in vitro, respectively. When serum was removed from the feeding medium after 9 days, the levels of these lipids did not increase and myelination failed to occur. When a 17 day explant was kept in medium containing [1-14C]lignoceric acid for 4 days, considerable radioactivity was taken up by the explant and incorporated into nonhydroxy- and hydroxycerebrosides, sulfatides and sphingomyelin. Most of the radioactivity in the alpha-hydroxy fatty acids was found in cerebronic acid, the product of lignoceric acid alpha-hydroxylation. Similar explants also took up [1-14C]palmitic acid when it was added to the medium. The radioactivity was, however, mostly incorporated into neutral lipids and glycerophospholipids. These observations indicate that the cultured mouse cerebellum explants synthesize and accumulate myelin-characteristic lipids as does brain in vivo.

Aging↗

PubMed1980

The metabolism of exogenous fatty acids by preimplantation mouse embryos developing in vitro.

The utilization of fatty acids from the culture medium has been examined in preimplantation mouse embryos developing in vitro. Incorporation of exogenous fatty acid into embryo lipids was examined by culturing 8-cell mouse embryos for 2h in a medium containing 0.1 mM [9, 10-3H]palmitic acid (900 mCi/mmol). Lipids were extracted from the embryos, and the total lipid extract was fractionated into various neutral lipid and polar lipid classes by thin-layer chromatography. Most of the radioactivity, over 93%, was recovered in neutral glycerides (mono- di-, and triacylglycerols). About 2% of the total radioactivity was recovered in other neutral lipid species including fatty acids, fatty alcohols, and sterol esters. The remainder of the radioactivity was recovered in polar lipids. Seventy-four per cent of the polar lipid radioactivity was present in the amine phosphatides, inositol and/or serine phosphatides, sphingomyelin, choline lysophosphatides, sulfatides, cerebrosides, and monoglycosylglycerides. Chemical degradation studies of labelled embryo lipids indicated that the tritium lable was entering into embryo lipids as the fatty acid and not via metabolic recycling. The oxidation of exogenous fatty acids by mouse embryos was assessed by incubating variously staged embryos for 4h in medium containing 0.1 mM [U-14C]palmitic acid (50 mCi/mmol) and quantitating the production of 14CO2. The rate of fatty acid oxidation was found to be relatively constant from the unfertilized egg up to the 8-cell stage and then increase significantly between the 8-cell and late blastocyst stages. The results suggest that preimplantation mouse embryos developing in vitro can utilize fatty acids from the medium both for incorporation into embryo lipids and for energy production via oxidation.

Animals↗

PubMed2002

Anti-obesity effect of dietary diacylglycerol in C57BL/6J mice: dietary diacylglycerol stimulates intestinal lipid metabolism.

We examined the long-term effects of dietary diacylglycerol (DG) and triacylglycerol (TG) with similar fatty acid compositions on the development of obesity in C57BL/6J mice. We also analyzed the expression of genes involved in lipid metabolism at an early stage of obesity development in these mice. Compared with mice fed the high-TG diet, mice fed the high-DG diet accumulated significantly less body fat during the 8-month study period. Within the first 10 days, dietary DG stimulated beta-oxidation and lipid metabolism-related gene expression, including acyl-CoA oxidase, medium-chain acyl-CoA dehydrogenase, and uncoupling protein-2 in the small intestine but not in the liver, skeletal muscle, or brown adipose tissue, suggesting the predominant contribution of intestinal lipid metabolism to the effects of DG. Furthermore, analysis of digestion products of [(14)C]DG and those of [(14)C]TG revealed that the radioactivity levels detected in fatty acid, 1-monoacylglycerol, and 1,3-DG in intestinal mucosa were significantly higher after intrajejunal injection of DG rather than TG. Thus, dietary DG reduces body weight gain that accompanies the stimulation of intestinal lipid metabolism, and these effects may be related to the characteristic metabolism of DG in the small intestine.

Animals↗

PubMed1981

Early changes in phosphatidylinositol and arachidonic acid metabolism in quiescent swiss 3T3 cells stimulated to divide by platelet-derived growth factor.

We added platelet-derived growth factor to cultures of quiescent Swiss 3T3 cells to investigate early changes in lipid metabolism related to initiation of cell cycle traverse. In a series of experiments that focused on lipid degradation we added the growth factor to cells that had been prelabeled with myoinositol, glycerol, or arachidonic acid. We observed the following mitogen-dependent effects: a decline of radioactivity in cell phosphatidylinositol within 2 to 5 min that progressed to 25 to 50% during the 1st h, a transient rise of radioactivity in cell diacylglycerol that peaked at 10 min, a gradual increase of radioactivity in monoacylglycerol in the medium, and a concomitant increase of radioactivity in medium-free fatty acid. In experiments that focused on lipid biosynthesis, we added the growth factor to cells and pulse-labeled them with radioactive precursors. We observed increased incorporation within 60 min of myoinositol into phosphatidylinositol, arachidonic acid into phosphatidylinositol, diacylglycerol, and phosphatidylethanolamine, and choline into phosphatidylcholine. These results support the possibility that action of platelet-derived growth factor on Swiss 3T3 cells leads to release of diacylglycerol from phosphatidylinositol, that some of the released diacylglycerol is hydrolyzed to monoacylglycerol and arachidonic acid, and that these lipid products are in part reconverted to phosphatidylinositol and other lipids.

Animals↗

PubMed1984

Comparison of lactate and glucose metabolism in the developing porcine placenta.

The present experiment was conducted to determine whether lactate is an important metabolic substrate for the developing porcine placenta. Pregnant gilts were anesthetized with pentobarbital sodium (5 mg/kg) at 65, 85, or 110 days of gestation and underwent an abdominohysterectomy. Fetal and maternal placental tissues were obtained and isolated for enzyme analysis and tissue incubations. Tissues were incubated for 2 h in Krebs-Ringer bicarbonate buffer containing 10 mM glucose, 1.0 mM Na-palmitate, 2.0 mM L-lactate, and 2% albumin. The incorporation of glucose or lactate into CO2, total lipids, and fatty acids was examined by radioactive tracers. Lactate was shown to be at least as important a substrate as glucose for utilization through these metabolic pathways in both the fetal and maternal placentas during gestation. This ability to metabolize lactate may serve as a conservation mechanism to spare other nutrients for transfer to the fetus. Additionally, analysis of enzyme and incubation data indicated that the fetal and maternal placenta follow different developmental patterns, implying the origins for (or mechanisms of) regulation of these two tissues may differ.

Animals↗

PubMed1990

Chain elongation of polyunsaturated fatty acids by vascular endothelial cells: studies with arachidonate analogues.

This study has utilized radiolabeled analogues of arachidonic acid to study the substrate specificity of elongation of long-chain polyunsaturated fatty acids. Human umbilical vein endothelial cells were incubated for 2-72 hr in medium supplemented with 0.9-2.6 microM [14C]fatty acid, and cellular glycerolipids were analyzed by gas-liquid chromatography with radioactivity detection. Elongation of naturally occurring C20 polyunsaturated fatty acids occurred with eicosapentaenoate (20:5(n-3] greater than Mead acid (20:3(n-9] greater than arachidonate (20:4(n-6]. Chain length markedly influenced the extent of elongation of 5,8,11,14-tetraenoates (18:4 greater than 19:4 greater than 20:4 greater than 21:4); effects of initial double bond position were also observed (6,9,12,15-20:4 greater than 4,7,10,13-20:4. Neither 5,8,14- nor 5,11,14-20:3 was elongated to the extent of 5,8,11-20:3. Differences between polyunsaturated fatty acids were observed both in the initial rates and in the maximal percentages of elongation, suggesting that the content of cellular C20 and C22 fatty acids may represent a balance between chain elongation and retroconversion. Umbilical vein endothelial cells do not exhibit significant desaturation of either 22:4(n-6) or 22:5(n-3). By contrast, incubation with 5,8,11,14-[14C]18:4(n-4) resulted in formation of both [14C]20:5(n-4) and [14C]22:5(n-4). The respective time courses for the appearances of [14C]22:5(n-4) and [14C]20:5(n-5) suggests delta 6 desaturation of [14C]22:4(n-4) rather than delta 4 desaturation of [14C]20:4(n-4).

8,11,14-Eicosatrienoic Acid↗