Sequestration of heat damaged rat erythrocytes during experimental action of methyl palmitate on RES

The effect of an i.v. injection of methyl palmitate emulsion (MP) on the clearance of heat damaged erythrocytes from the blood and their sequestration in organs of rats was followed. Twenty-four hours following application of MP in the dose of 1.5 g/kg of body weight the survival half time of erythrocytes (T1/2(51)Cr) significantly increased, whereas the amount of sequestrated red cells in the spleen decreased. The subsequent intervals of examination, however, showed no differences as compared to the controls.     

The effect of intravenous injection of ethyl palmitate emulsion on sequestration of thermally damaged erythrocytes in rats with experimentally induced hypersplenism, and in normal rats.

The authors attempted at experimental elimination of sequestration function of the spleen in Wistar rats using an i.v. injection of ethyl palmitate emulsion, both in "hypersplenic" animals being long-term applied i.p. methyl cellulose solution, and in control rats. In the rats clearance of 51Cr-labelled and thermally damaged erythrocytes from blood was examined and their sequestration in the spleen and liver followed. The ethyl palmitate injection resulted in both experimental groups in a significant decrease of the erythrocyte counts sequestrated in the spleen, and significant prolongation of the elimination half time for thermally damaged erythrocytes from the blood.     

The inhibitory effect of methyl palmitate on heteroagglutinin formation in rats.

In Wistar rats the immunosuppressive effect of methyl palmitate on the formation of heteroagglutinins against human O-group erythrocytes was followed. An i.v. injection of methyl palmitate both delayed the heteroagglutinin formation and decreased its intensity. The inhibitory effect of methyl palmitate was not accompained by the subsequent hyperactive phase as could be observed in the previous experiments using ethyl palmitate.     

Characterization of chylomicron remnant clearance by retinyl palmitate label in normal humans.

To characterize chylomicron remnant clearance by the liver, plasma elimination of retinyl palmitate-labeled chylomicron remnants was studied in 18 healthy subjects, ages 21-42 years. Autologous plasma containing retinyl palmitate-labeled chylomicrons and their remnants was injected intravenously, and retinyl palmitate disappearance was measured in serial plasma samples in all subjects and in lipoprotein fractions in 11 subjects. The injected doses (n = 18) ranged from 0.34 to 7.11 mumol retinyl palmitate in d less than or equal to 1.006 g/ml particles with an average molar ratio of 330/1 of retinyl palmitate/apoB-48 (n = 8). The label distributed in the intravascular space and exhibited apparent first order elimination, monoexponential in 6 and biexponential in 12 subjects. The first rapid component k1 (t1/2 18.8 +/- 11.4 min, n = 18) was shown to represent retinyl palmitate in particles of d less than or equal to 1.006 g/ml, i.e., chylomicron remnants, and the second slow component k2 (t1/2 123 +/- 62 min, n = 12) small amounts of retinyl palmitate (11 +/- 7%) injected in d greater than 1.006 g/ml particles (therefore excluded from analysis). Assuming a single-compartment model, initial rates of elimination (= dose x k1) of labeled chylomicron remnants obeyed (P = 0.06) Michaelis-Menten saturation kinetics: Km was 921 +/- 305 nmol retinyl palmitate label and Vmax 124 +/- 14 nmol/min corresponding to 0.88 nM apoB-48 for Km and 0.25 x 10(-3) nmol apoB-48.min-1.g-1 liver for Vmax. Their elimination was limited neither by the injected triglyceride dose nor theoretically by the liver blood flow. After the intake of 70 g of fat (cream) containing retinyl palmitate, the plasma retinyl palmitate concentration exceeded the estimated saturation concentration for 7 h. In conclusion, physiological chylomicron remnant catabolism by the liver appears to be saturable by ordinary lipid intake in healthy humans.     

Methyl palmitate inhibits lipopolysaccharide-stimulated phagocytic activity of rat peritoneal macrophages

Macrophages, in general, are critical effectors of body's immune system. Chemical inhibition of phagocytic activity of such macrophages as Kupffer cells has been extensively studied. We have earlier shown that methyl palmitate (MP) inhibits the activation of Kupffer cells. To evaluate the potential of MP to inhibit the activation of other macrophages, we treated rat peritoneal macrophages with varying concentrations of MP. Its treatment led to a dose-dependent inhibition of phagocytic activity, which was found to be 34%, 47%, and 66% at 0.25, 0.50, and 1.0 mM MP, respectively, as measured by latex bead uptake. When MP-treated peritoneal macrophages were stimulated with lipopolysaccharide (LPS), the nitric oxide (.NO) release was inhibited at 6 h, while cyclooxygenase-2 expression decreased after 24 h. The treatment with MP increased the release of interleukin (IL)-10 in the LPS-treated cells at 6 h, while IL-6 and tumor necrosis factor-alpha were significantly increased both at 6 and 24 h. Our data suggest that MP inhibits phagocytic activity and .NO production similar to that observed in isolated Kupffer cells. Therefore, inhibition of phagocytosis by MP may be a general phenomenon, and it could be used as an inhibitor of macrophage function.     

Comparison of linoleate,palmitate and acetate metabolism in rat ventral prostate

Rat ventral prostate incorporated (1-¹⁴C)acetate, (1-¹⁴C)palmitate and (1-¹⁴C)linoleate into different phospholipids in a time-dependent process. The rate of incorporation into total phospholipids was higher with linoleate (10.0 nmol/g) than with either palmitate (5.8 nmol/g) or acetate (4.7 nmol/g). Predominant labelling with all the radioactive substrates assayed was found in choline glycerophospholipids (PC). The radioactive profiles for linoleate in the other ventral prostate phospholipids differed from those obtained with palmitate and acetate. Specifically linoleate was incorporated into inositol glycerophospholipids plus lysoethanolamine glycerophospholipids (PI+LPE) and not into sphingomyelin (SM), while palmitate and acetate incorporated into SM but not into PI+LPE. Acetate showed the highest oxidation to CO₂ whereas no differences were observed in the radioactivity incorporated into CO₂ from a saturated (palmitate) or an essential unsaturated fatty acid (linoleate). These studies also show zinc-dependence by the acetate to CO₂ oxidation.Abbreviations PL total phospholipids - PC choline glycerophospholipids - PE ethanolamine glycerophospholipids - PI+LPE inositol glycerophospholipids plus lysoethanolamine glycerophospholipids - PS serine glycerophospholipids - SM sphingomyelin     

Parameters affecting productivity in the lipase-catalysed synthesis of sucrose palmitate

The industrial application of lipases for the synthesis of sucrose esters is usually limited by its low productivity, as we need a medium where a polar reagent (the sugar) and a non-polar fatty acid donor are soluble and able to react in the presence of the biocatalyst. In this work, we have studied the problems encountered when trying to increase the volumetric productivity of sucrose esters. The synthesis of sucrose palmitate was performed in 2-methyl-2-butanol:dimethylsulfoxide mixtures by transesterification of different palmitic acid donors with sucrose, catalysed by the immobilized lipase from Candida antarctica B (Novozym 435). A protocol for substrate preparation different from that previously reported was found to improve the reaction rate. Several parameters, such as sucrose and acyl donor loadings, the percentage of DMSO in the mixture and the nature of acyl donor, were investigated. Under the best experimental conditions (15% DMSO, 0.1 mol l^?1 sucrose, 0.3 mol l^?1 vinyl palmitate), a maximum of 45 g l^?1 sucrose palmitate was obtained in 120 h. Using methyl or ethyl palmitate, the highest productivity was 7.3 g l^?1 in 120 h using 20% DMSO with 0.2 mol l^?1 sucrose and 0.6 mol l^?1 acyl donor. The formation of free fatty acid, and the effect of the percentage of DMSO on the monoester/diester selectivity were also studied. To our knowledge, this is the first report on enzymatic synthesis of sucrose esters of long fatty acids using alkyl esters as acyl donors.     

Muscle palmitate uptake and binding are saturable and inhibited by antibodies to FABPPM

Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABP_PM) may be a component of this system. To test the hypothesis that FABP_PM is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABP_PM. Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated V_max values of 10.5 ± 1.2 pmol/mg protein/15 sec and 45.6 ± 2.9 nmol/mg protein/15 min and K_m values of 12.8 ± 3.8 and 18.4 ± 1.8 nmol/L, respectively. The V_max values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABP_PM. Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABP_PM. Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABP_PM is a component of this process in muscle.     

Influence of starvation on the lung: effect on glucose and palmitate utilization.

The relative utilization of [U-14C]glucose and [1-14C]palmitate was examined in lung slices of male Long Evans hooded rats fed ad libitum and starved for 72 h. Food deprivation (72-h fast) significantly decreased [U-14C]flucose oxidation and incorporation into lung lipids. Glucose incorporation into phospholipid-fatty acid (53%) was, in proportion, more markedly reduced than into phospholipid-gluceride glycerol (33%), suggesting that glucose was being conserved for the formation of alpha-glycerol phosphate. (1-14C) palmitate utilization following fasting showed a significant 40% increase in oxidation, and a significant 16% increase in phospholipids, indicating preferential utilization of fatty acids over glucose. Phospholipid fatty acid composition, surface tension measurements and volume-pressure curves were not affected by fasting. Khe data indicate that glucose and palmitate metabolism are interrelated, and that the relative utilization of these substrates is changed to maintain essential lung lipids during an altered physiologic state.     

Methyl palmitate: a novel product of the Medfly (Ceratitis capitata) corpus allatum

The corpus allatum (CA) of adult female Ceratitis capitata produces methyl palmitate (MP) in vitro, in addition to JHB₃ and JH III. Biosynthesized MP migrates on TLC and co-elutes from RP-18 HPLC with synthetic MP. Its identity is verified herein by GCMS. MP production is up-regulated twofold by mevastatin, an inhibitor of mevalonic acid-dependent isoprene biosynthesis. Fosmidomycin, an inhibitor of mevalonic acid-independent isoprene synthesis in graminaceous plants, up-regulates MP synthesis by about fourfold. However, it does not depress JHB₃ biosynthesis concurrently. This suggests that the initial enzyme(s) in the conversion of 1-deoxy-xylulose 5-phosphate to isoprene is presumably present in C. capitata, but is inhibited by fosmidomycin, and this inhibition diverts precursors to MP synthesis. Phytol, an acyclic diterpene, might be suppressing isoprene biosynthesis by CA, thereby resulting in a fourfold increase in the MP biosynthesis. Linolenic acid is an end-product and its presence in incubation media up-regulates MP biosynthesis by twofold, presumably due to the feedback diversion to biosynthesis of C_16:0 and its methyl ester. Biosynthesis of MP is markedly depressed after mating, while otherwise maintained at significantly higher levels in virgin females. MP biosynthesis is significantly reduced in virgin females by direct axonal control but is less consistent after mating.     

Molecular mechanism by which palmitate inhibits PKR autophosphorylation

PKR (double-stranded RNA-activated protein kinase) is an important component of the innate immunity, antiviral, and apoptotic pathways. Recently, our group found that palmitate, a saturated fatty acid, is involved in apoptosis by reducing the autophosphorylation of PKR at the Thr451 residue; however, the molecular mechanism by which palmitate reduces PKR autophosphorylation is not known. Thus, we investigated how palmitate affects the phosphorylation of the PKR protein at the molecular and biophysical levels. Biochemical and computational studies show that palmitate binds to PKR, near the ATP-binding site, thereby inhibiting its autophosphorylation at Thr451 and Thr446. Mutation studies suggest that Lys296 and Asp432 in the ATP-binding site on the PKR protein are important for palmitate binding. We further confirmed that palmitate also interacts with other kinases, due to the conserved ATP-binding site. A better understanding of how palmitate interacts with the PKR protein, as well as other kinases, could shed light onto possible mechanisms by which palmitate mediates kinase signaling pathways that could have implications on the efficacy of current drug therapies that target kinases.     

Presence and titer of methyl palmitate in the Medfly (Ceratitis capitata) during reproductive maturation

The relative amounts of methyl palmitate (MP) during the first 10 days post-eclosion were determined in whole-body extracts of adult female Ceratitis capitata by SIM monitoring of the 74 m/z fragment. MP peaks in receptive 3-day-old virgin females coincide with previously reported production of Juvenile Hormone (JH) by the corpus allatum (CA). Mating in the Medfly induces female non-receptivity. Indirect evidence suggests that the mevalonate pathway to sesquiterpene biosynthesis is underdeveloped in newly eclosed females. We propose that the pathway leading to synthesis of JH is markedly diverted in non-receptive virgin females to fatty acid synthesis, and partly so-in non-receptive mated females, leading to production of palmitic acid, presumably methylated thereafter. MP is depressed and remains marginal thereafter for the 7 days examined in the virgin female but goes through an apparent second cycle in the mated female. This contrasts with the consistent increase of allatal biosynthesis of MP of virgin and mated females previously reported and suggests additional control mechanisms in vivo. During the period of reduced receptivity following the first mating a second apparent peak of MP is observed. MP is a metabolic default metabolite of reproductively immature females whose putative role in reproductive physiology remains to be defined.     

Is there an optimal haematocrit for rainbow trout,Oncorhynchm mykiss (Walbaum)? An interpretation of recent data based on blood viscosity measurements

The viscosity of blood from rainbow trout was measured following manipulation of haematocrit by bleeding, hypoxia. exercise, and anaesthesia. Blood viscosity when measured at high shear rate (225 s ¹) was proportional to haematocrit, but the dependence of viscosity on shear rate was far less for swollen erythrocytes from exercised and anaesthetized trout. Erythrocyte swelling was most marked in exercised and anaesthetized trout, and is a confounding factor when considering the effect of haematocrit on viscosity.
The viscosity of blood with variable haematocrit, but constant mean cell Hb concentration, indicated that the relative oxygen transport capacity in trout was optimal at a haematocrit of 30%. Data from this, and earlier studies show that haematocrit in trout is variable and labile, yet none of the haematocrit values following manipulations are less than 85% of optimal. Optimal haematocrit is however, significantly higher than measured values from either cannulated or acutely venesected resting trout.     

Training-induced elevation in FABP(PM) is associated with increased palmitate use in contracting muscle.

To evaluate the effects of endurance training in rats on fatty acid metabolism, we measured the uptake and oxidation of palmitate in isolated rat hindquarters as well as the content of fatty acid-binding proteins in the plasma membranes (FABP(PM)) of red and white muscles from 16 trained (T) and 18 untrained (UT) rats. Hindquarters were perfused with 6 mM glucose, 1,800 microM palmitate, and [1-(14)C]palmitate at rest and during electrical stimulation (ES) for 25 min. FABP(PM) content was 43-226% higher in red than in white muscles and was increased by 55% in red muscles after training. A positive correlation was found to exist between succinate dehydrogenase activity and FABP(PM) content in muscle. Palmitate uptake increased by 64-73% from rest to ES in both T and UT and was 48-57% higher in T than UT both at rest (39.8 +/- 3.5 vs. 26.9 +/- 4. 4 nmol. min(-1). g(-1), T and UT, respectively) and during ES (69.0 +/- 6.1 vs. 43.9 +/- 4.4 nmol. min(-1). g(-1), T and UT, respectively). While the rats were resting, palmitate oxidation was not affected by training; palmitate oxidation during ES was higher in T than UT rats (14.8 +/- 1.3 vs. 9.3 +/- 1.9 nmol. min(-1). g(-1), T and UT, respectively). In conclusion, endurance training increases 1) plasma free fatty acid (FFA) uptake in resting and contracting perfused muscle, 2) plasma FFA oxidation in contracting perfused muscle, and 3) FABP(PM) content in red muscles. These results suggest that an increased number of these putative plasma membrane fatty acid transporters may be available in the trained muscle and may be implicated in the regulation of plasma FFA metabolism in skeletal muscle.     

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.     

Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.     

Starvation-induced changes of palmitate metabolism and insulin secretion in isolated rat islets stimulated by glucose.

The influence of 48 h starvation on glucose-induced changes of palmitate metabolism and insulin release in isolated rat islets was investigated. (1) Islet insulin response to 20 mM-glucose was abolished after 48 h starvation, and it was restored by 0.25 mM-2-bromostearate, an inhibitor of fatty acid oxidation. (2) The increase in glucose concentration from 3 to 20 mM was accompanied by a 50% decrease in the oxidation rate of 0.5 mM-[U-14C]palmitate in control (fed) islets, and a concomitant increase (100%) in its incorporation into triacylglycerol and phospholipid fractions. (3) Starvation induced a higher basal (3 mM-glucose) rate of palmitate oxidation, which was resistant to inhibition by 20 mM-glucose. The latter also failed to increase palmitate incorporation into islet triacylglycerols and phospholipids. (4) 2-Bromostearate (0.25 mM) strongly inhibited the high oxidation rate of palmitate in islets of starved rats, and allowed a normal stimulation of its incorporation rate into islet lipids by 20mM-glucose. (5) The results suggest that starvation restricts islet esterification of fatty acids by inducing a higher rate of their oxidative degradation that is insensitive to regulation by glucose.     

Transport and metabolism of palmitate in the rat liver. Net flux and unidirectional fluxes across the cell membrane.

The unidirectional fluxes of palmitate across the liver cell membrane and metabolic uptake rates were measured employing the multiple-indicator dilution technique. The following results were obtained: (1) Influx and net uptake rates do not vary proportionally to each other when albumin and palmitate concentrations are varied. (2) Efflux is significant for albumin concentrations in the range between 1.5 and 500 microM. (3) At 150 microM albumin net uptake rates are proportional to the total (bound plus free) extracellular palmitate concentration in the range from 10 to 600 microM; the dependence of influx rates on the palmitate concentration is rather concave up. (4) When albumin and palmitate are both varied at an equimolar ratio, pseudo-saturation appears in the net uptake rates; the influx rates also show pseudo-saturation, but with a declining tendency at the higher concentrations. (5) The intracellular palmitate concentration is strongly influenced by albumin. At very low concentrations of the protein (1.5 microM) the intracellular concentration is practically equal to the extracellular one; at physiological albumin concentrations, however, the intracellular palmitate concentration is less than 2% of the extracellular one. (6) Saturation of net uptake with respect to the intracellular palmitate concentration was not observed with concentrations up to 46 microM.     

Regulation of fetal lung disaturated phosphatidylcholine synthesis by de novo palmitate supply

Lung surfactant disaturated phosphatidylcholine (PC) is highly dependent on the supply of palmitate as a source of fatty acid. The purpose of this study was to investigate the importance of de novo fatty acid synthesis in the regulation of disaturated PC production during late prenatal lung development. Choline incorporation into disaturated PC and the rate of de novo fatty acid synthesis was determined by the relative incorporation of [14C]choline and 3H2O, respectively, in 20-day-old fetal rat lung explants and in 18-day-old explants which were cultured 2 days. Addition of exogenous palmitate (0.15 mM) increased (26%) choline incorporation into disaturated PC but did not inhibit de novo fatty acid synthesis, as classically seen in other lipogenic tissue. Even in the presence of exogenous palmitate, de novo synthesis accounted for 87% of the acyl groups for disaturated PC. Inhibition of fatty acid synthesis by agaric acid or levo-hydroxycitrate decreased the rate of choline incorporation into disaturated PC. When explants were subjected to both exogenous palmitate and 60% inhibition of de novo synthesis, disaturated PC synthesis was below control values and 75% of disaturated PC acyl moieties were still provided by de novo synthesis. These data show that surfactant disaturated PC synthesis is highly dependent on the supply of palmitate from de novo fatty acid synthesis.     

Decreased hepatic retinyl palmitate hydrolase activity in protein-deficient rats

28-day-old weanling rats were fed a diet containing 3% casein as the only source of protein for eight weeks to induce protein deficiency. When compared to control animals (fed a diet containing 25% casein), these rats had significantly lowered body (5.2-fold reduction) and liver (2.5-fold reduction) weights. The circulatory level of retinol (nmol per ml plasma) as well as retinol (nmol per g tissue) in the liver of these protein-deficient animals were also reduced significantly, although their liver concentration of retinyl palmitate (nmol per g tissue) was comparable to that of the control group. Assay of liver tissue for retinyl palmitate hydrolase activity revealed a 4-fold reduction (compared to that of control animals) of specific enzyme activity (nmol retinol formed per g protein per h). These findings suggest that severe protein deficiency results in a decreased hydrolysis of retinyl esters in the liver, which may be in part responsible for the reduced level of metabolically 'active' retinoids available for normal physiological functions.