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[The spectrum of fatty acids during lactation in women].

The large spectrum of fatty acids (expressed in relative values) was studied in the breast-human milk during the first three weeks of the lactation (3rd, 5th, 10th and 20th day after delivery). All women passed the pregnancy and the delivery without any pathological syndrome and their neonates were found as a healthy and physiological too. No drug dependency was registered. Fatty acids were measured as methyl-esters (FAME) using the method of gas chromatography (1). During the 20 days after delivery and of lactation period, summarising the groups of fatty acid (saturated, monoenoic, PUFA n-3, n-6 and n-9) only few changes could be detected: the increase of the participation of saturated fatty acids of short-medium chain (8:0, 10:0 and 12:0) and of PUFA n-9. Compared with the most administered commercial formula Feminar, the visible differences in the participation of fatty acids were described. Finally: during the lactation we could obtain a large number of significant differences of such individual fatty acids as palmitic acid, arachidonic acid, eicosopentaenoic acid, linoleic acid ect. The possible reasons of such changes during the lactation are discussed and these results are compared with our previous data concerning the fatty acid- spectrum in blood plasma of healthy newborns and preterm or hypoxic infants.

Fatty Acids↗

[Identification and characterization of hydrophobic ligand binding region in glutathione S-transferase P].

Recombinant glutathione S-transferase P (GST-P) was purified in a homogeneous state. Fatty acid analysis by gas-liquid chromatography-mass spectrometry (GC-MS) revealed that GST-P forms 1:1 complex with fatty acids, mostly palmitic acid or stearic acid, which were hardly isolated from the complex even through Lipidex 1,000 column chromatography at 37 degrees C. Temperature dependent analysis of 1H-NMR on the association between GST-P and fatty acids indicated that molecular motion of fatty acids were strongly restrained in a hydrophobic 'pocket' below the temperature of protein denaturation. On the other hand, there existed another hydrophobic ligand binding region, to which fatty acid and bilirubin would bind with relatively lower affinity. The binding region was determined to be at around 142-157 residues from amino terminus by the studies of GST-P binding to fatty acid-linked Sepharose and affinity labelings with either fluorescent fatty acid or bilirubin. The binding to this region noncompetitively inhibited the enzyme activity. Furthermore, circular dichroism (CD) analysis showed that the binding of hydrophobic ligands changed the secondary structure of GST-P, which suggested that the enzyme activity was regulated through conformational changes. As tryptophan 38 was assumed to locate at the active center from the study of site-directed mutagenesis, conformation of the active center was investigated by measuring the intrinsic tryptophan fluorescence. It showed that hydrophobic ligand binding caused the drastic conformational change, of which would be referred to the regulation of the enzyme activity.

Binding Sites↗

Relations between vitamin D and fatty acid binding properties of vitamin D-binding protein.

The interaction of fatty acids with bovine vitamin D-binding protein (DBP) was studied using a partition equilibrium method. This protein has one high affinity site for binding of fatty acids with an association constant Ka = 7 x 10(5) M-1 for palmitic acid and Ka = 6 x 10(5) M-1 for arachidonic acid. Competition experiments showed that palmitic acid hardly competes with 25-hydroxycholecalciferol for binding to DBP. However, arachidonic acid showed comparatively a stronger competition for binding to this protein. The great difference in competition of palmitic and arachidonic acids with 25-hydroxycholecalciferol may be related to changes in DBP conformation promoted by the binding of different ligands.

Animals↗

Arachidonic acid causes cell death through the mitochondrial permeability transition. Implications for tumor necrosis factor-alpha aopototic signaling.

We have investigated the effects of arachidonic and palmitic acids in isolated rat liver mitochondria and in rat hepatoma MH1C1 cells. We show that both compounds induce the mitochondrial permeability transition (PT). At variance from palmitic acid, however, arachidonic acid causes a PT at concentrations that do not cause PT-independent depolarization or respiratory inhibition, suggesting a specific effect on the PT pore. When added to intact MH1C1 cells, arachidonic acid but not palmitic acid caused a mitochondrial PT in situ that was accompanied by cytochrome c release and rapidly followed by cell death. All these effects of arachidonic acid could be prevented by cyclosporin A but not by the phospholipase A(2) inhibitor aristolochic acid. In contrast, tumor necrosis factor alpha caused phospholipid hydrolysis, induction of the PT, cytochrome c release, and cell death that could be inhibited by both cyclosporin A and aristolochic acid. These findings suggest that arachidonic acid produced by cytosolic phospholipase A(2) may be a mediator of tumor necrosis factor alpha cytotoxicity in situ through induction of the mitochondrial PT.

Animals↗

Hydrolysis of 11-cis- and all-trans-retinyl palmitate by retinal pigment epithelium microsomes.

A partial characterization of the enzymatic hydrolysis of 11-cis- and all-trans-retinyl palmitate by bovine retinal pigment epithelium microsomes was carried out using a micro-radiometric method to quantitate liberated palmitic acid. Retinyl ester hydrolase (REH) activity was examined in the absence of detergent. Hydrolysis of 11-cis- and all-trans-retinyl palmitate was protein- and time-dependent. Optimal enzyme activity occurred at slightly alkaline pH (8-9). Apparent kinetic constants (Vmax and Km) for the 11-cis-REH were 2.1 nmol/min/mg protein and 66 microM, respectively. All-trans-REH demonstrated a lower maximum velocity of 0.3 nmol/min/mg protein and a slightly higher substrate affinity of 27 microM. Further characterization of 11-cis-retinyl palmitate hydrolysis involved monitoring formation of reaction products, 11-cis retinol and palmitic acid, which were found to be released in essentially a 1:1 stoichiometry. Addition of all-trans retinyl bromoacetate, a known inhibitor of lecithin:retinol acyltransferase reduced both 11-cis and all-trans-REH activities but to significantly different degrees (50 and 76%, respectively). Although the microsomal preparation exhibited LRAT activity, acyl transfer was not readily reversible as labeled palmitic acid was not transferred to added acyl acceptor compounds. These findings suggest that hydrolysis of 11-cis-retinyl palmitate by bovine retinal pigment epithelium microsomes may occur at a catalytic site distinct from that for the all-trans isomer and that this hydrolysis is not representative of a reverse transesterification reaction.

Animals↗

Cytoplasmic lipid bodies of human neutrophilic leukocytes.

The morphology and function of cytoplasmic lipid bodies in human neutrophils were evaluated. By transmission electron microscopy, neutrophil lipid bodies were cytoplasmic inclusions, usually several microns in diameter, that occasionally coalesced to attain a diameter up to 7 microM. Neutrophil lipid bodies were not enveloped by membrane but were often surrounded by a more electron-dense shell at their periphery. Normal peripheral blood neutrophils contained an average of approximately one lipid body per cell. Lipid bodies appeared in greater numbers in neutrophils from inflammatory lesions. Perturbation of neutrophils during conventional methods of cell isolation and purification modestly increased lipid body numbers in neutrophils, whereas incubation of neutrophils with 1 microM oleic acid rapidly induced lipid body formation over 30 to 60 minutes. After granulocytes were incubated for 2 hours with 3H-fatty acids, including arachidonic, oleic, and palmitic acids, electron microscopic autoradiography demonstrated that lipid bodies represented the predominant intracellular sites of localization of each of the three 3H-fatty acids. There was lesser labeling noted in the perinuclear cisterna, but not in cell membranes. Virtually all of each of the three 3H-fatty acids incorporated by the neutrophils were esterified into chromatographically resolved classes of neutral lipids or phospholipids. These findings indicate that cytoplasmic lipid bodies are more prominent in neutrophils in vivo engaged in inflammatory responses and that these organelles in human neutrophils function as sites of deposition of esterified, incorporated fatty acids.

Arachidonic Acid↗

Carboxyatractylate inhibits the potentiating effect of lipophylic cation TPP+ on uncoupling activity of fatty acid.

The effect of TPP+ on the fatty acid or FCCP-induced uncoupling in rat heart mitochondria was studied. It was found that (a) TPP+ increases the stimulation of oxygen consumption by palmitic acid or FCCP in the presence of oligomycin, (b) TPP+ greatly enhances the palmitic acid or FCCP-induced delta psi decrease. Both effects of TPP+ were strongly suppressed by carboxyatractylate in the case of palmitate but were not in the case of FCCP. The role of ATP/ADP-antiporter in the TPP+ and palmitic acid effects is discussed.

Animals↗

The metabotropic glutamate receptor mGluR4, but not mGluR1 alpha, is palmitoylated when expressed in BHK cells.

Several G protein-coupled receptors have been shown to be palmitoylated, and for some of these receptors the covalent attachment of palmitate has been implicated in the regulation of receptor-G protein coupling. The metabotropic glutamate receptor (mGluR) family forms a distinct group of G protein-coupled receptors, and the possibility that these may also be palmitoylated has been examined. Clonal baby hamster kidney (BHK) cells permanently transfected with the mGluR4 and mGluR1 alpha subtypes were labelled with [3H]palmitic acid. The cells were lysed, the receptors were immunoprecipitated with specific antipeptide antibodies, and the immunoprecipitates were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. The palmitoylated, endogenously expressed G protein alpha-subunit alpha q could be immunoprecipitated from [3H]palmitate-labelled BHK cells expressing mGluR1 alpha using a specific antipeptide antibody, but in the same cell lysates no detectable [3H]palmitate-labelled mGluR1 alpha was found. This suggests that this mGluR subtype, associated with stimulation of phospholipase C, is not palmitoylated. In contrast, mGluR4, which is coupled to inhibition of adenylyl cyclase, was found to be labelled with [3H]palmitic acid, and the palmitate was quantitatively removed by treatment with 1 M hydroxylamine, suggesting attachment of the palmitate through a thioester bond. Stimulation with maximal doses of the neurotransmitter glutamate for 1, 5, or 10 min appeared to have no effect on the level of receptor palmitoylation.

Animals↗

Qualitative and quantitative changes in milk fat during lactation in the tammar wallaby (Macropus eugenii).

There are major quantitative and qualitative changes in the milk lipids during lactation in the tammar wallaby, Macropus eugenii. The crude lipid content of the milk is relatively low during the first 10 weeks of lactation; between 10 and 26 weeks post partum the lipid content increases gradually but after that it increases rapidly. The triglyceride fraction of the lipid at early stages of lactation contains a large amount of palmitic acid and relatively little oleic acid whereas mature milk exhibits little palmitic and much oleic acid. In the early stages of lactation fat represents 15% of the total solids and carbohydrate 55%; around 26-30 weeks post partum the carbohydrate moiety falls sharply to a level less than 2% of the solids while lipids increase to c. 60% of the solids. These changes coincide with increases in milk solids, emergence of the young from the pouch, ingestion of herbage, and fermentation of cellulose in the stomach.

Animals↗

Stable isotope dilution method for measurement of palmitate content and labeled palmitate tracer enrichment in microliter plasma samples.

In studies where [1-13C]palmitic acid is employed as isotopic tracer in vivo, we have described a selected ion monitoring gas-liquid chromatography-mass spectrometry micro-method which allows plasma tracer enrichment as well as plasma palmitate content to be determined in the same 100-microliter sample through the use of [5,5,6,6-2H4]palmitic acid as assay internal standard. For standard solutions in the range equivalent to plasma palmitic acid concentrations of 10--2500 microM, assay precision was +/- 5%. For plasma samples in the physiologic range (approximately 30--200 microM palmitate) assay precision averaged better than +/- 2%. The use of the method is illustrated by measuring palmitate turnover in a 12-hr-old human infant.

Carbon Isotopes↗

In vitro acylation of rat brain myelin proteolipid protein.

The incorporation of fatty acid into myelin proteolipid protein (PLP) was studied by incubating brain slices from 17-day-old rats with [9,10-3H]palmitic acid. Myelin proteins were analyzed by gel electrophoresis. As in vivo (Agrawal, H. C., Randle, C. L., and Agrawal, D. (1982) J. Biol. Chem. 257, 4588-4592) most of the radioactivity co-migrated with PLP, with radioactivity also appearing in the regions of the DM-20 and Wolfgram proteins. Treatment of myelin proteins or purified PLP with 1 M hydroxylamine released most of the radioactivity, indicating that [3H]palmitic acid was covalently bound by ester linkage to the proteins. Cleavage of PLP with methanolic NaOH and examination of the released fatty acids by thin layer chromatography showed radioactivity associated with the methyl esters of palmitate, stearate, and oleate (76, 23, and 1%, respectively). A linear increase in radioactivity from [3H]palmitic acid was observed in PLP in both myelin and myelin-like fractions, while appearance of [14C]glycine-labeled PLP showed a lag of 45-60 minutes. These results indicate that lipid is added to PLP in the myelin and myelin-like membranes, or immediately prior to the appearance of PLP in these fractions. For both precursors, the specific activity of PLP was greater in the myelin-like fraction than in myelin, indicating that the myelin-like fraction may contain a precursor pool of PLP for myelin.

Acylation↗

In vitro fenoprofenyl-coenzyme A thioester formation: interspecies variations.

In vitro coenzyme A thioester formation from (-)-(R)-fenoprofen (FPF) and palmitic acid has been studied using liver microsomes from rat, guinea pig, sheep, and dog. In every species with both palmitic acid or (-)-(R)-fenoprofen, the Lineweaver-Burk plot was linear in the substrate concentration range used and as a consequence agrees with the involvement of only one isoenzyme (or different isoenzymes of similar Km values). The Vmax values for the thioesterification of (-)-(R)-fenoprofen present large species variations from 2.1 +/- 1.0 with sheep liver microsomes to 60.6 +/- 11 nmol/min/mg with dog liver microsomes. These values statistically significantly correlate (r = 0.94) to the Vmax values observed when palmitic acid was used as a substrate. Furthermore palmitic acid inhibited (-)-(R)-fenoprofen-CoA formation in the same extent in all animal species. The stereoselectivity of the thioesterification was also species dependent.

Animals↗

Insulin response to glucose-6-phosphate dehydrogenase activity is elevated in rats fed diets low in polyunsaturated fatty acids.

Glucose-6-phosphate dehydrogenase (G6PD) activity in the rat liver was elevated when the animals were fed a diet that contained 5% coconut oil or 1% corn oil plus 4% palmitic acid, in comparison with that of rats fed 5% corn oil. G6PD activity correlated inversely with the PUFA/SFA ratio of total liver phospholipid as well as the ratio of plasma membrane phospholipid. Elevation of G6PD activity was slightly affected by dietary protein. Serum insulin levels were apparently not influenced by dietary fats, and were not correlated with G6PD activity. Insulin dose-response to G6PD activity was augmented in primary cultured hepatocytes isolated from rats fed a diet with 1% corn oil plus 4% palmitic acid in comparison with those fed a diet with 1% corn oil plus 4% palmitic acid in comparison with those fed a diet with 5% corn oil. These findings indicate that augment in insulin dose-response to G6PD and elevation of its activity shown in rats fed diets low in polyunsaturated fatty acid are associated with lowering the PUFA/SFA ratio in plasma membrane phospholipid.

Animals↗

The regulation of fatty acid chain elongation in rat liver microsomes: role of fasting and CoASH.

The elongation system for palmitic acid in rat liver microsomes was decreased to 1/3 by fasting, while the elongation of eicosapentaenoic acid was not sensitive to fasting. The rate of eicosapentaenoic acid elongation in the fed state was 50% higher than using palmitic acid as a substrate. The saturated and polyunsaturated fatty acyl-CoA substrates exhibited positive cooperativity on the rate-limiting condensing step in the elongation system, with a Hill constant of approx. 2. An inhibition by CoASH on the total elongation reaction as well as on the condensation step was demonstrated using acyl-CoA substrates, and followed a hyperbolic pattern. The concentrations giving a 50% inhibition (30-70 microM) were in the range found in rat hepatocyte cytosol, indicating that free CoASH has the potential to act as a physiological regulator.

Animals↗

Effects of fatty acids on Na/Ca exchange in cardiac sarcolemmal membranes.

Three structurally distinct amphiphiles palmitic acid, oleic acid, and palmityl carnitine were studied to determine their effects on sodium dependent calcium uptake by purified cardiac sarcolemmal vesicles (PSL). Sodium dependent calcium uptake by PSL when studied over a 20 min reaction period was composed of an initial rapid uptake (20.9 +/- 0.93 nmol/mg X 30 s, mean +/- S.E. n = 20) a plateau in calcium content (42.4 +/- 3.2 nmol/mg, mean +/- S.E. n = 20) and a slow spontaneous release characterized by a first order rate constant of 0.68 +/- 0.08/h (mean +/- S.E. n = 18). Both palmityl carnitine and palmitic acid inhibited, whereas oleic acid stimulated initial calcium uptake. All three amphiphiles shortened the time to peak calcium content, inhibited peak calcium content and increased the rate constant for calcium release. All these effects were observed at fatty acid: membrane phospholipid mole ratios of 0.67 : 1 to 1.67 : 1 for oleic acid and palmityl carnitine and 0.02 : 1 to 0.42 : 1 for palmitic acid. These effects do not reflect disruption of membrane vesicle structure and may be explained, at least in part, by amphiphile induced increases in sarcolemmal membrane ion permeability. Although amphiphile accumulation has been implicated in the pathogenesis of cellular abnormalities in the ischemic myocardium, this study has shown that large amounts of amphiphile relative to membrane lipid are required to alter sarcolemmal membrane function in vitro.

Animals↗

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

Specific differences between the acyl composition of lipids of the helminth Paratenuisentis ambiguus and its host eel, as shown previously, prompted us to study the lipid metabolism in this intestinal fish parasite. Adults and larvae of P. 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 in P. ambiguus at a high rate to labeled palmitic acid, which was incorporated into various lipid classes of P. 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 ether-glycerophospholipids of P. 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 of P. ambiguus with [1-14C]palmitic or [1-14C]oleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Acanthocephala↗

Fatty acid requirement of Treponema denticola and Treponema vincentii.

Treponema denticola and Treponema vincentii were cultured in a medium supplemented with either 0.2 or 0.4% (w/v) alpha globulin in place of serum. The active factor(s) in alpha globulin was stable at pH 7.0 to autoclaving and was nondialyzable. Extraction of lipids from alpha globulin showed that both protein and lipid, supplied by the alpha globulin, were required for maximal growth of these two oral treponemes. The lipid component was investigated by adding sodium salts of long-chain fatty acids to the basal medium supplemented with 0.4% delipified alpha globulin. The lipid component of alpha globulin was replaced by either oleic acid (cis-18:1(9)) or by elaidic acid (trans- 18:1 (9)0. No other saturated or unsaturated fatty acid tested could support good growth. Tween 80 (polysorbitan monooleate) was the only Tween compound able to support maximal growth of T. denticola. The cellular lipids of T. denticola, grown with oleate in broth supplemented with 0.4% delipified alpha globulin, were extracted and analyzed by gas chromatography. The principle fatty acids were myristic, pentadecanoic, and palmitic acids. Lesser amounts of oleic acid, eicosadienoic acid, and an unidentified fatty acid (retention time, 88 min) were also detected. Treponema denticola appears to be capable of limited synthesis of cellular fatty acids such as myristic, pentadecanoic, and palmitic acids from oleic acid.

Alpha-Globulins↗

Effects of monensin on posttranslational processing of myelin proteins.

Rat brain slices were incubated with [3H]palmitic acid and [14C]glycine to label the lipid and protein moieties, respectively, of myelin proteolipid protein (PLP). The effects of monensin on posttranslational processing of proteins were examined by measuring the appearance of [14C]glycine- and [3H]palmitate-labeled proteins in myelin and myelin-like fractions. At 0.01 and 0.10 microM, monensin did not appreciably affect total lipid or protein synthesis; higher concentrations caused increased inhibition. Monensin at 0.10 microM markedly decreased the appearance of [14C]glycine-labeled PLP in myelin, but had little effect on the 14C basic proteins or the incorporation of [3H]palmitic acid into total or myelin PLP. The same relative effect was apparent at higher monensin concentrations. In the myelin-like fraction, monensin at 0.10 microM also depressed entry of [14C]glycine into protein comigrating with PLP, and again had no effect on incorporation of [3H]palmitic acid. In addition, monensin increased the [3H]palmitate label associated with two high-molecular-weight proteins in the myelin-like fraction with no concomitant increase in [14C]glycine label.

Animals↗