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Effect of alpha-ketobutyrate on palmitic acid and pyruvate metabolism in isolated rat hepatocytes.

alpha-Ketobutyrate, an intermediate in the catabolism of threonine and methionine, is metabolized to CO2 and propionyl-CoA. Recent studies have suggested that propionyl-CoA may interfere with normal hepatic oxidative metabolism. Based on these observations, the present study examined the effect of alpha-ketobutyrate on palmitic acid and pyruvate metabolism in hepatocytes isolated from fed rats. alpha-Ketobutyrate (10 mM) inhibited the oxidation of palmitic acid by 34%. In the presence of 10 mM carnitine, the inhibition of palmitic acid oxidation by alpha-ketobutyrate was reduced to 21%. These observations are similar to those previously reported using propionate as an inhibitor of fatty acid oxidation, suggesting that propionyl-CoA may be responsible for the inhibition. alpha-Ketobutyrate (10 mM) inhibited 14CO2 generation from [14C]pyruvate by more than 75%. This inhibition was quantitatively larger than seen with equal concentrations of propionate. Carnitine (10 mM) had no effect on the inhibition of pyruvate oxidation by alpha-ketobutyrate despite the generation of large amounts of propionylcarnitine during the incubation. alpha-Ketobutyrate inhibited [14C]glucose formation from [14C]pyruvate by more than 60%. This contrasted to a 30% inhibition caused by propionate. These results suggest that alpha-ketobutyrate inhibits hepatic pyruvate metabolism by a mechanism independent of propionyl-CoA formation. The present study demonstrates that tissue accumulation of alpha-ketobutyrate may lead to disruption of normal cellular metabolism. Additionally, the production of propionyl-CoA from alpha-ketobutyrate is associated with increased generation of propionylcarnitine. These observations provide further evidence that organic acid accumulation associated with a number of disease states may result in interference with normal hepatic metabolism and increased carnitine requirements.

Acyl Coenzyme A↗

Effects of ammonium chloride, salicylate, and carnitine on palmitic acid oxidation in rat liver slices.

To explore the possible association of hyperlipidemia with hyperammonemia and aspirin ingestion, the effects of NH4+, salicylate, and carnitine on the oxidation of [1-14C]palmitic acid to acid-soluble products (ASP) and to CO2 were investigated in rat liver slices. DL-carnitine (5 mM) increased total oxidation (ASP + CO2) more than oxidation to CO2. KCN (1.5 mM) inhibited more than 90% of the oxidation. NH4Cl inhibited the oxidation that reached a maximum at about 40 mM, but the inhibition of oxidation to CO2 (63%) was larger than that of total oxidation (30%). Carnitine did not influence NH4+ inhibition, which is consistent with the results reported for isolated mitochondria. Salicylate effects depended on salicylate concentration as well as on the presence of carnitine. In the absence of carnitine, inhibition of total oxidation reached 90% at 3 mM salicylate but that of oxidation to CO2 reached 50%. Velocity calculated at saturating palmitic acid concentration for total oxidation was slightly increased by 0.75 mM salicylate, but the increase for oxidation to CO2 was larger. At 3 mM salicylate, velocity at saturating palmitic acid concentration for the oxidation was decreased, but the decrease for oxidation to CO2 was smaller than for total oxidation. Carnitine partially relieved the inhibition of total oxidation and further increased the formation of CO2. The combination of 20 mM NH4Cl and 0.75 mM salicylate inhibited total oxidation, which was more than additive of the individual effects, and carnitine partially relieved the inhibition. It is concluded that NH4+ exerted a stronger inhibition of oxidation to CO2 than of oxidation to ASP, whereas salicylate strongly inhibited the oxidation to ASP but increased the oxidation to CO2 by uncoupling mitochondrial oxidative phosphorylation. Therefore, hyperammonemia and aspirin ingestion can inhibit fatty acid oxidation and mitochondrial metabolism that could lead to the pathophysiology seen in some childhood diseases such as Reye's syndrome. Carnitine therapy might offer some benefits.

Ammonium Chloride↗

[1-(11)C]palmitic acid: improved radiopharmaceutical preparation.

A simple synthetic procedure and processing system for the routine preparation of [1-(11)C]palmitic acid via carbonation of n-pentadecylmagnesium bromide with [11C]carbon dioxide is described. The key feature of this improved synthesis is the facile separation of the 11C fatty acid from the radiolabeled and unlabeled by-products of the Grignard reaction. Using this procedure and system 7.40-9.25 GBq (200-250 mCi) of [11C]CO2 was converted, after a synthesis time of 30 min, into 1.85-2.78 GBq (50-75 mCi) of pure, sterile, pyrogen-free [1-(11)C]palmitic acid suitable for human use with positron computed tomography.

Carbon Radioisotopes↗

Palmitic acid but not stearic acid inhibits NO-production in endothelial cells.

Several studies showed a diminished production of the endothelium-derived relaxing factor nitric oxide (NO) in the early stage of atherosclerosis. The inhibition of NO-production seems to be mediated by lipoproteins, especially oxidized low-density lipoproteins (ox-LDL). There is some evidence, that the interactions of lipoproteins and NO are associated with the phospholipid fraction of lipoproteins. Since fatty acids have different atherogenic properties-depending on chain length, degree of saturation and steric configuration-, we investigated the effect of fatty acids on endothelial NO-production. Human umbilical vein endothelial cells were incubated with palmitic acid and stearic acid in different concentrations in culture medium enriched with serum albumin for five hours. After that, NO-production was stimulated by calcium-ionophore A23187. NO-production was determined by a bioassay method using RFL-6 cells followed by radioimmunological determination of cGMP. NO-production stimulated by calcium-ionophore A23187(100%) was decreased by palmitic acid (10, 50, 100 microM) to 79 +/- 12%; 63 +/- 10% and 53 +/- 14%. In contrast, incubation with stearic acid (10, 50 and 100 microM) had no effect on A23187-stimulated NO-production (94 +/- 11%; 93 +/- 11%; 104 +/- 15%). Thus, palmitic acid but not stearic acid dose-dependently inhibited NO-release by endothelial cells. These different actions parallel the differing atherogenic potential of the two fatty acids.

Calcimycin↗

Acylation of cell-associated IL-1 by palmitic acid.

To determine whether membrane-associated IL-1 is palmitylated, we labeled LPS-activated human monocytes with [3H]palmitic acid. The plasma membranes were isolated, and the membrane proteins extracted and analyzed simultaneously by SDS-PAGE-autoradiography and Western blot analysis from the same gel. When the monocytes were labeled with [3H]palmitate, 23- and 31-kDa bands were visualized, for membrane-associated IL-1 and its precursor, respectively. The 31- and 23-kDa bands were excised from several gels and rehydrated and analyzed again by SDS-PAGE, autoradiography, and Western blot analysis. The 23- and 31-kDa bands appeared again by both methods. To further investigate membrane-associated IL-1 acylation, human monocytes were labeled with [3H]palmitate, the plasma membranes isolated, and the membrane proteins extracted by CHAPS detergent. Immunoprecipitation of isolated membrane proteins using anti-IL-1 antibodies revealed two bands of 23 and 31 kDa after autoradiography. The studies demonstrate that both membrane-associated IL-1 and the IL-1 precursor are acylated with palmitic acid.

Acylation↗

[The effect of temperature on lysis of human erythrocytes by palmitic acid].

An analysis of kinetic curves of erythrocyte hemolysis induced by palmitic acid has shown the existence of some stages of this process. The activation energy of hemolysis, as determined by the temperature dependence of the hemolysis rate constant, was 210 +/- 30 kJ/mol. It was shown by the method of stepwise thermoinactivation of erythrocytes proteins that at temperature of 49 degrees C which corresponded to the framework protein spectrin denaturation temperature, the erythrocyte membrane stability sharply decreased. On the contrary, changes of the cell shape induced by the hyperosmotic medium (0.5 M sucrose) inhibited the palmitic acid-induced erythrocytes hemolysis.

Erythrocyte Membrane↗

[Is the intake of palm oil (palmitic acid) in meals associated with the low incidence of respiratory distress syndrome in Nigeria? (author's transl)].

Comparative gaschromatographic assays of different domestic oils used in West Germany and the domestic oil (palm oil) used in Nigeria and other West African countries were performed. It was discovered that the palm oil contains extremely high amounts of palmitic acid and linoleic acid compared to the other oils used in Germany. In view of the fact that previous studies have shown that palmitic acid is required for the biosynthesis of lung lecithin, which is closely related to fetal maturation, we performed animal experiments with rabbits to determine the incorporation rate of 3H-marked palmitic acid into various fetal organs: (fetal lung, placenta, fetal liver, fetal intestine etc.) after a previous intravenous injection of the 3H-marked palmitic acid to the mother rabbit. The radiochromatogram showed high incorporation rates of palmitate into the lecithin by fetal lung and placenta. These results seem to document that the high content of palm oil in Nigerian meals can partly be related to the low incidence of respiratory distress syndrom in Nigerian premature infants.

Animals↗

Dipalmitoylphosphatidylcholine-palmitic acid phase diagram studied by 13C nuclear magnetic resonance.

The phase diagram of dipalmitoylphosphatidylcholine (DPPC) and palmitic acid mixtures in excess D2O was studied by 13C-NMR. Phase boundaries were determined from plots of apparent spin-spin relaxation time T2 (for both choline methyl and fatty acid chain carbons) versus temperature. A peritectic transition in the 1-10 mol% region, whose existence has been theoretically inferred from the Gibbs phase rule but which was undetectable by differential thermal analysis (DTA) (S.E. Schullery et al. Biochemistry, 20 (1981) 6818-6824), was located by NMR at 41.6 degrees C. A second, nearby peritectic line at 44 degrees C, which had been shown by DTA to extend from about 3-25 mol% palmitic acid, was seen by NMR only above 10 mol%. The palmitic acid/DPPC complex (2:1), with a sharp melting point at 64 degrees C, reported in earlier studies, was also seen by NMR. A phase diagram including both NMR and DTA results is presented. Important general conclusions from this study are: (i) NMR and scanning thermal analysis are complementary techniques for phase studies; each can see transitions that are invisible to the other. (ii) The case for the applicability of the Gibbs phase rule to lipid bilayer systems has been strengthened by the observance of two predicted, close-spaced boundaries. (iii) Low concentrations of fatty acids and related molecules can not be assumed to disperse as simple ideal solutes in the bilayer matrix.

Carbon Isotopes↗

Selective use of palmitic acid over stearic acid for synthesis of phosphatidylcholine and phosphatidylglycerol in lung.

The incorporation of [3H]palmitic acid and [14C]stearic acid into phospholipids in rabbit lung tissue was studied. Under equal molar concentrations of palmitate and stearate, palmitate was incorporated to the 1- and 2-positions of phosphatidylcholine (PC) and phosphatidylglycerol (PG) 2-3 times more than stearate. By contrast, palmitate was 30% less than stearate in phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine. These results suggest that preferential utilization of palmitate over stearate, rather than substrate availability, determines the high content of palmitoyl at the 1- and 2-positions of PC and PG in lung.

Animals↗

Synthesis and use of stereospecifically deuterated analogues of palmitic Acid to investigate the stereochemical course of the delta11 desaturase of the processionary moth.

Thaumetopoea pityocampa pheromone glands contain desaturases that, after several sequential reactions from palmitic acid, catalyze the formation of a unique enyne fatty acid, which is the immediate sex pheromone precursor. In this article, we describe the synthesis of different stereospecifically deuterium-labeled and isotopically tagged palmitic acid probes needed to decipher the stereochemical course of the T. pityocampa Delta(11) desaturase. The synthesis of probes has been carried out by a chemoenzymatic route, in which the key step is the kinetic lipase-catalyzed resolution of racemic mixtures of secondary propargyl alcohols. The presence of the acetylenic bond simplifies the absolute configuration determination of the resolved alcohols. Moreover, it allows the introduction of the isotopic tag by deuteration. By use of the probes thus prepared, experimental evidence is presented that the Delta(11) desaturase of T. pityocampa transforms palmitic acid into (Z)-11-hexadecenoic acid by removal of the pro-(R)-hydrogen atoms from both C11 and C12.

Animals↗

Characterization of water-soluble products of palmitic acid beta-oxidation by a rat brain preparation.

(1) [1-(14)C]Palmitic acid was oxidized to CO(2) and a water-soluble material by a rat brain preparation. The radioactive CO(2) and water-soluble material were produced in a ratio of 1.0:1.3 when the mitochondrial fraction was used, and 1.0:10 or more with the postnuclear fraction. There was a lag period of 10 min for CO(2) production. These conversions were stimulated by carnitine and inhibited by cyanide. (2) Of the total radioactivity in the water-soluble material obtained with the mitochondrial fraction, 65% after 10 min of incubation and 80% thereafter were associated with amino acids, mostly with aspartate and glutamate. The remaining radioactivity, 35 and 20%, respectively, was associated with organic acids, 60-65% in citrate. The water-soluble material obtained with the postnuclear fraction contained an equal amount of radioactivity in organic and amino acids during the course of the experiment. In the organic acids, succinate was the highest labeled product during 10-40 min of incubation, whereas citrate was the highest labeled at the end of 60 min of incubation. After 60 min, the radioactivity in the amino acids was markedly associated with glutamate, and its radioactivity was 10 times greater with the postnuclear fraction than with the mitochondrial one. (3) An experiment with rat live preparations was also carried out. The liver mitochondrial fraction showed an accumulation of radioactive organic acids within 10 min of incubation, which was followed by a linear production of (14)CO(2). With the liver postnuclear fraction, the radioactivity was found mostly in the organic acids during the course of the experiment. In the liver system, the radioactive amino acids accounted for only 25% or less of the total radioactivity in the water-soluble material.

3-Hydroxybutyric Acid↗

Amniotic fluid palmitic acid concentrations and prediction of fetal lung maturity.

A method for rapid determination of total esterified palmitic acid concentration (TEPAC) in amniotic fluid is described. The correlation coefficient between the TEPAC and the lecithin concentration was 0.93 in 123 samples of amniotic fluid obtained during the last trimester. The respiratory distress syndrome (RDS) occurred in 73% of the cases studied with TEPAC less than mmol/l in predelivery samples. The RDS was not observed with higher concentrations except in cases of maternal diabetes mellitus. The predictive value of total esterified fatty acid studies was confirmed to the concentration of palmitic acid recorded, and no specific fatty acid distributions or ratios were reliable in identifying RDS or estimating gestational age.

Amniotic Fluid↗

Effects of palmitic acid and palmityl carnitine on calcium sequestration by rabbit skeletal sarcoplasmic reticulum vesicles.

A number of long-chain fatty acids and fatty acid derivatives accumulate in the cytosol of ischemic myocardium. Although the functional significance of this accumulation in vivo remains unclear, these amphiphilic compounds may alter the functional properties of a variety of biological membranes in vitro. In this study, we investigated the effect of palmitic acid and palmityl carnitine on calcium sequestration by sarcoplasmic reticulum vesicles in the absence of calcium-precipitating anions. Palmitic acid, at micromolar concentrations, enhanced calcium sequestration in a concentration-dependent manner when present from the onset of the reaction or when added to calcium-filled vesicles. Under identical conditions, similar concentrations of palmityl carnitine inhibited calcium sequestration when present at the onset of the reaction and caused a rapid release of accumulated calcium when added to calcium-filled vesicles. Low concentrations of palmitic acid decreased the sensitivity of the sarcoplasmic reticulum to the inhibitory effects of palmityl carnitine. These results suggest that calcium pump function in the sarcoplasmic reticulum can be altered by the presence of amphiphilic compounds and that this alteration is dependent on both the structure and number of amphiphiles present.

Animals↗

The palmitic acid binding properties of cytosolic proteins located in the villus and crypt zones of bovine intestinal mucosa.

The palmitic acid binding capacity of cytosolic proteins in three preparations obtained by differential scraping of bovine intestinal mucosa were compared. The data indicated that the palmitic acid binding activities depended on the position that the cells occupied along the crypt-villus axis, as shown from the level of alkaline phosphatase activity. Proteins with palmitate binding properties in the high- and low-molecular-weight cytosolic proteins in the villus zone bound 1.24 +/- 0.41 and 1.54 +/- 0.16 pmol palminate/micrograms protein respectively. The binding decreased to 0.50 +/- 0.25 and 1.10 +/- 0.23 pmol palmitate/micrograms for the proteins in the crypt zone. Ammonium sulphate fractionation and gel filtration chromatography indicated that the low-molecular-weight cytosolic proteins obtained from light mucosal scrapings contained the highest palmitate binding activity. These results suggest that the cytosolic proteins located in the villus zone may play a role in the absorption of fatty acids.

Alkaline Phosphatase↗

Pulmonary lung surfactant synthetic peptide concentration-dependent modulation of DPPC and POPG acyl chain order in a DPPC:POPG:palmitic acid lipid mixture.

Lung surfactant-associated protein interaction with lipid matrices and the effects on lipid thermotropic phase behavior are areas of active research. Many studies limit the lipids to a single or two-component system. The current investigation utilizes a three-lipid component matrix (DPPC:POPG:palmitic acid) to investigate the impact of a synthetic surfactant protein B fragment (SP-B 53-78 DiACM) on the dynamic surface activity of the lipid admixture as measured by a Wilhelmy surface balance. Also, the modulation of the individual lipid acyl chain order by the peptide within the lipid matrix is studied through the use of thermal perturbation FTIR spectroscopy. The data clearly demonstrate a concentration-dependent effect of the peptide on the surface activity with an improvement in the dynamic surface tension diagram characteristics (decreased surface tension and increased collapse plateau) especially at low, 0.36 M%, peptide concentrations. These effects are diminished upon further addition of the peptide. FTIR spectral data demonstrate that the peptide addition results in a significant increase in the acyl chain order of the DPPC and POPG components as measured by the position of the methylene stretching vibrational bands. DPPC is most sensitive to the peptide presence, while the palmitic acid is least affected. The transition temperatures of the individual lipids are also increased with the addition of the peptide. The presence of POPG in the matrix achieves the surface activity similarly seen with natural lung surfactant relative to a DPPC/palmitic acid lipid matrix alone. Its presence increases the sensitivity of the DPPC acyl chains to the presence of the peptide. These effects on the chain order are most probably related to the increased acyl chain fluidity which POPG imparts to the lipid matrix because of the presence of the cis double bond. The phosphatidylglycerol headgroup also adds a negative charge to the lipid matrix which enhances the peptide-lipid interaction. Although the palmitic acid is minimally affected by the peptide, its presence, as suggested by surface balance measurements, results in the establishment of a stable lipid film with DPPC, capable of achieving low surface tension values.

1,2-Dipalmitoylphosphatidylcholine↗

Incorporation of palmitic acid or oleic acid into macrophage membrane lipids exerts differential effects on the function of normal mouse peritoneal macrophages.

Normal mouse peritoneal macrophages were enriched with either palmitic acid (16:0) or oleic acid (18:1). Normal or oleic acid-enriched macrophages showed 3-4-fold greater erythrophagocytic capacity as compared to palmitic acid-enriched macrophages. Staphylococcus aureus uptake was only moderately decreased in palmitic acid-enriched macrophages. Fatty acid modifications did not influence the ability of macrophages to kill intracellular bacteria or to generate superoxide anions after stimulation with phorbol myristate acetate or opsonized zymosan.

Animals↗

Isolation and properties of a particulate fraction for the desaturation of palmitic acid from Alcaligenes faecalis.

A particulate fraction obtained from Alcaligenes faecalis could desaturate palmitic acid to palmitoleic acid. NADPH, ATP, CoA, Fe2+ and Mg2+ were essential cofactors for the reaction. The desaturation showed an absolute requirement for O2. Metal ions like Mn2+, Mo6+ and Cu2+ did not affect the desaturation, while Zn2+ was inhibitory. Sulfhydryl agents such as cysteine, glutathione and beta-mercaptoethanol had no effect, but SH-blocking agents like HgCl2 and p-hydroxymercuribenzoate inhibited the reaction. Azide and cyanide strongly inhibited the reaction while CO had no effect. The presence of a b-type cytochrome in the enzyme preparation was confirmed by the spectral studies on the reaction of enzyme with NADPH. Involvement of b-type cytochrome in the desaturation reaction was demonstrated by the reoxidation of b-type cytochrome initially reduced with NADPH, by the addition of palmitic acid and other cofactors. The pH optimum for the enzyme activity was 7.4. The optimum temperature for enzyme activity was 25 degrees C and maximum activity was obtained at the end of 45 min.

Adenosine Triphosphate↗