Carmem Maldonado Peres

Toxicity of a Soybean Oil Emulsion on Human Lymphocytes and Neutrophils

BACKGROUND The incorporation of lipid emulsions in parenteral diets is a requirement for energy and essential fatty acid supply to critically ill patients. In this study, the toxicity of a lipid emulsion rich (60%) in triacylglycerol of omega-6 polyunsaturated fatty acids on leukocytes from healthy volunteers was investigated. METHODS Eleven volunteers were recruited, and blood samples were collected before infusion of a soybean oil emulsion, immediately afterwards, and 18 hours later. The cells were studied immediately after isolation and again after 24 hours or 48 hours in culture. The following determinations were made: composition and concentration of fatty acids in plasma, lymphocytes and neutrophils, lymphocyte proliferation, levels of cell viability, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, reactive oxygen species production, and neutral lipid accumulation. RESULTS Soybean oil emulsion decreased lymphocyte proliferation and provoked neutrophil and lymphocyte apoptosis and necrosis. Evidence is presented herein that soybean oil emulsion is less toxic to neutrophils than to lymphocytes. The mechanism of cell death induced by this oil emulsion was characterized by mitochondrial membrane depolarization and neutral lipid accumulation but did not alter reactive oxygen species production. CONCLUSIONS Soybean oil emulsion given as a single dose of 500 mL promotes lymphocyte and neutrophil death that may enhance the susceptibility of the patients to infections.

Neutrophil Death Induced by a Triathlon Competition in Elite Athletes

INTRODUCTION/PURPOSE The effect of a triathlon competition on death of neutrophils from elite athletes was investigated. METHODS Blood was collected from 11 sedentary volunteers and 12 triathletes under rest and after a Half Ironman triathlon competition (2-km swimming, 80-km cycling, and 20-km running). RESULTS The triathlon competition increased DNA fragmentation, phosphatidylserine externalization, and reactive oxygen species production in neutrophils when compared to the results at rest. The proportion of neutrophils with mitochondrial transmembrane depolarization was increased in the triathletes at rest and after competition as compared with sedentary volunteers. Plasma levels of thiobarbituric acid reactive substances were increased in triathletes after competition. Expression of bcl-xL (antiapoptotic) was decreased and that of bax (proapoptotic) was increased, whereas intracellular neutral lipid content was lowered in neutrophils after the triathlon. A positive correlation was found between the proportion of neutrophils with DNA fragmentation and the plasma free fatty acid levels (r = 0.688, P< 0.05), which was elevated by threefold after competition. Plasma levels of oleic, linoleic, and stearic acids were increased in triathletes after the competition when compared with sedentary volunteers. The plasma concentration of these three fatty acids, measured after the triathlon competition, was toxic for 3-h cultured neutrophils obtained from sedentary volunteers. The maximal tolerable (nontoxic) concentration of the fatty acids by 3-h cultured neutrophils was 100 micromol x L(-1) for oleic and linoleic acids and 200 micromol x L(-1) for stearic acid. CONCLUSION The triathlon competition induced neutrophil death possibly by apoptosis as indicated by DNA fragmentation and phosphatidylserine externalization. The increase in plasma levels of oleic, linoleic, and stearic acids induced by the competition may be involved in the neutrophil death observed possibly by increasing the production of reactive oxygen species and by decreasing the accumulation of intracellular neutral lipid.

Effect of fish oil supplementation for two generations on changes of lymphocyte function induced by Walker 256 cancer cachexia in rats.

Fish oil supplementation has been shown to improve the cachectic state of tumor-bearing animals and humans. Our previous study showed that fish oil supplementation (1 g per kg body weight per day) for 2 generations had anticancer and anticachetic effects in Walker 256 tumor-bearing rats as demonstrated by reduced tumor growth and body weight loss and increased food intake and survival. In this study, the effect of fish oil supplementation for 2 generations on membrane integrity, proliferation capacity, and CD4/CD8 ratio of lymphocytes isolated from mesenteric lymph nodes, spleen, and thymus of Walker 256 tumor-bearing animals was investigated. We also determined fish oil effect on plasma concentration and ex vivo production of cytokines [tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-6, and IL-10]. Lymphocytes from thymus of tumor-bearing rats presented lower viability, but this change was abolished by fish oil supplementation. Tumor growth increased proliferation of lymphocytes from all lymphoid organs, and fish oil supplementation abolished this effect. Ex vivo production of TNF-α and IL-6 was reduced in supplemented animals, but IL-4 and IL-10 secretion was stimulated in both nontumor and tumor-bearing rats. IL-10 and IFN-γ plasma levels was also decreased in supplemented animals. These results suggest that the anticachetic effects of fish oil supplementation for a long period of time (2 generations) in Walker 256 tumor-bearing rats may be associated to a decrease in lymphocyte function as demonstrated by reduced viability, proliferation capacity, and cytokine production.

Effect of lipid infusion on metabolism and force of rat skeletal muscles during intense contractions.

The hypothesis that during intense muscle contraction induced by electrical stimulation, long chain fatty acids (LCFA) might reduce mitochondrial ATP/ADP ratio, raising the contribution of glycolysis for ATP production was examined. The effect of a lipid infusion (Lipovenus emulsion) on UCP-3 mRNA level, lactate, glucose-6-phosphate (G-6P) and glycogen content was investigated in rat. Blood samples for determination of free fatty acids and lactate were collected at 0, 30 and 60 min during rest and at 0, 10 and 20 min during muscle contraction. The content of lactate, glycogen and G-6P was also determined in soleus (SO), red gastrocnemius (RG) and white gastrocnemius (WG) muscles collected immediately after muscle contraction period. In addition, the force level was determined during muscle contractions. The effect of Lipovenus emulsion on respiration of mitochondria isolated from rat skeletal muscle, and content of UCP-3 and lactate in cultured skeletal muscle cells was also determined. The in vivo experiments showed that Lipovenus induced a significant increase of UCP-3 mRNA levels. After Lipovenus infusion, lactate level was increased in RG muscle only, whereas the contents of glycogen and G-6P were decreased in both RG and WG muscles (P < 0.05). Lipovenus infusion failed to exert any effect on muscle force performance (P > 0.05). The in vitro experiments showed that Lipovenus infusion induced a significant increase in mitochondrial respiration, but had no effect on UCP-3 content. Lactate concentration was significantly increased in the culture medium of stimulated cells in the control and Lipovenus groups compared with the respective not-stimulated cells (P< 0.05). We concluded that as mitochondrial function becomes limited by the FFA-uncoupling effect, the ATP demand is mainly supplied by anaerobic glucose metabolism preventing an expected decrease in muscle contraction performance.

Thioglycolate-elicited rat macrophages exhibit alterations in incroporation and oxidation of fatty acids

Incorporation and oxidation of fatty acids (FA) were investigated in resident and thioglycolate-clicited (TG-elicited) rat macrophages (Mϕ). Both cell types presented a time-dependent incorporation of [14C]-labeled palmitic acid (PA), oleic acid (OA), linoleic acid (LA), and arachidonic acid (AA) up to 6h. The total amount of [14C]-FA incorporated by resident Mϕ after 6 h was: AA>PA=LA>OA. TG-elicited cells presented a 50% reduction in the incorporation of LA, PA, and AA, whereas that of OA remained unchanged as compared to resident Mϕ. The FA were oxidized by resident Mϕ as follows: LA>OA>PA>AA. TG elicitation promoted a reduction of 42% in LA oxidation and a marked increase in AA oxidation (280%). The increased oxidation of AA in TG-elicited cells may account for the lower production of prostaglandins in Mϕ under these conditions. The full significance of these findings for Mϕ function, however, remains to be examined.

Leukocyte infiltration in lung, muscle, and liver after limb compression in rats

Muscle crush injury is associated with systemic manifestations known as crush syndrome. A systemic inflammatory response syndrome may be triggered by isolated crush injury. Using myeloperoxidase (MPO) activity and plasma fatty acid composition, we investigated the inflammatory response in distant organs after isolated limb compression in rats. Male Wistar rats were submitted to 1h of hind limb compression by a latex ribbon. Myeloperoxidase activity was measured in muscle, liver, and lung at progressive times (1, 2 or 4h) after bandage release. Plasma fatty acid composition was evaluated as an indirect measure of oxidative stress. The liver and hind limb muscles showed a transient increase in MPO activity. Pulmonary MPO activity, otherwise, increased progressively throughout the study and reached statistically significant values at 4h when compared to all other groups (p<0.05). Plasma levels of unsaturated fatty acids decreased gradually after decompression (p<0.05 compared to controls after 4h). Blunt traumatic muscle compression was associated with rapid and transient muscle and liver inflammatory cell infiltration but otherwise, polymorphonuclear cells showed progressive aggregation in lungs. The plasmatic unsaturated index decreased throughout the 4h after muscle release. We demonstrated that limb compression was associated with oxidative stress and distant inflammatory responses. Progressive inflammatory cell infiltration in lungs could be related with the delayed systemic adverse responses found after crush injury.

Lonomia obliqua (Lepidoptera, Saturniidae) caterpillar bristle extract induces direct lysis by cleaving erythrocyte membrane glycoproteins.

Lonomia obliqua caterpillar bristle extract induces hemolysis in vitro on washed human and rat erythrocytes, in either the absence or presence of exogenous lecithin. In the former condition, phospholipases A(2) are key enzymes involved in hemolysis. However, the mechanism whereby this extract causes direct hemolysis is not known. Thus, the aim of this study was to investigate the hemolytic mechanism of the crude extract of the caterpillar L. obliqua on human erythrocytes in the absence of lecithin. The extract significantly increased the erythrocyte osmotic fragility and promoted the removal of glycophorins A and C, and band 3 from the erythrocyte membrane. The use of Ca2+ and Mg2+ ions significantly potentiated glycoprotein removal, remarkably of erythrocyte band 3. The composition of fatty acids was analyzed by HPLC in both L. obliqua caterpillar bristle extract and human erythrocyte membranes incubated with the extract. The levels of unsaturated fatty acids were remarkably augmented in erythrocytes incubated with the extract than in control erythrocytes, modifying thereby the saturated/unsaturated fatty acid ratio. Altogether, evidence is provided here that the interplay of at least three mechanisms of action accounts for the direct activity of the bristle extract on erythrocyte membrane, leading to hemolysis: the removal of glycoproteins and band 3; the insertion of fatty acids; and the action of phospholipases. Such mechanisms might affect erythrocyte flexibility and deformability, which may induce hemolysis by increasing erythrocyte fragility. However, whether the direct hemolytic activity of L. obliqua caterpillar is the major cause of intravascular hemolysis during envenomation still needs further investigation.

Fatty acid transport across lipid bilayer planar membranes

The transport of palmitic acid (PA) across planar lipid bilayer membranes was measured using a high specific activity [14C]palmitate as tracer for PA. An all-glass trans chamber was employed in order to minimize adsorbance of PA onto the surface. Electrically neutral (diphytanoyl phosphatidylcholine) and charged (Azolectin) planar bilayers were maintained at open electric circuit. We found a permeability to PA of (8.8±1.9)×10−6 cm s−1 (n=15) in neutral and of (10.3±2.2)×10−6 cm s−1 (n=5) in charged bilayers. These values fall within the order of magnitude of those calculated from desorption constants of PA in different vesicular systems. Differences between data obtained from planar and vesicular systems are discussed in terms of the role of solvent, radius of curvature, and pH changes.

Transfer of arachidonic acid from lymphocytes to macrophages

The incorporation and oxidation of arachidonic acid (AA) by rat lymphocytes (LY), the transfer of AA from LY to rat macrophages (Mϕ) in co-culture, and the subsequent functional impact on Mϕ phagocytosis were investigated. The rate of incorporation of [1-14C]AA by untreated-LY and TG (thioglycolate treated)-LY (TG-LY) was 158±8 nmol/1010 LY per h for both untreated-LY and TG-LY. The oxidation of AA was 3.4-fold higher in TG-LY as compared with untreated cells. LY from TG-injected rats had a 2.5-fold increase in the oxidation of palmitic (PA), oleic (OA), and linoleic (LA) acids. After 6 h of incubation, [14C] from AA was distributed mainly into phospholipids. The rate of incorporation into total lipids was 1071 nmol/1010 cells in untreated-LY and 636 nmol/1010 cells in TG-LY. [14C]AA was transferred from LY to co-cultured Mϕ in substantial amounts (8.7 nmol for untreated and 15 nmol per 1010 for TG cells). Exogenously added AA, PA, OA, and LA caused a significant reduction of phagocytosis by resident cells. Mϕ co-cultured with AA-preloaded LY showed a significant reduction of the phagocytic capacity (about 40% at 35 μM). LY preloaded with PA, LA, and OA also induced a reduction in phagocytic capacity of co-cultured Mϕ. TG treatment abolished the AA-induced inhibition of phagocytosis in Mϕ co-cultured with TG-LY. Therefore, the transfer of AA between leukocytes is a modulated process and may play an important role in controlling inflammatory and immune response.

Plasma cytokine expression after lower-limb compression in rats ☆

Objectives Muscle injury due to crushing (muscle compression injury) is associated with systemic manifestations known as crush syndrome. A systemic inflammatory reaction may also be triggered by isolated muscle injury. The aim of this study was to investigate the plasma levels of interleukins (IL) 1, 6 and 10 and tumor necrosis factor alpha (TNF-α), which are markers for possible systemic inflammatory reactions, after isolated muscle injury resulting from lower-limb compression in rats. Methods Male Wistar rats were subjected to 1 h of compression of their lower limbs by means of a rubber band. The plasma levels of IL 1, 6 and 10 and TNF-α were measured 1, 2 and 4 h after the rats were released from compression. Results The plasma levels of IL 10 decreased in relation to those of the other groups, with a statistically significant difference (p < 0.05). The method used did not detect the presence of IL 1, IL 6 or TNF-α. Conclusion Our results demonstrated that the changes in plasma levels of IL 10 that were found may have been a sign of the presence of circulating interleukins in this model of lower-limb compression in rats.