Inhibition of rat microsomal lipid peroxidation by the oral administration of D002

The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS). When D002 (5-100 mg/kg body weight) was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46%) occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg) also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40%) and brain (28-44%) microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg) for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ß-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ß-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ß-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ß-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ß-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

Inhibition of in vitro CO2 production and lipid synthesis by 2-hydroxybutyric acid in rat brain

2-Hydroxybutyric acid appears at high concentrations in situations related to deficient energy metabolism (e.g., birth asphyxia) and also in inherited metabolic diseases affecting the central nervous system during neonatal development, such as "cerebral" lactic acidosis, glutaric aciduria type II, dihydrolipoyl dehydrogenase (E3) deficiency, and propionic acidemia. The present study was carried out to determine the effect of 2-hydroxybutyric acid at various concentrations (1-10 mM) on CO2 production and lipid synthesis from labeled substrates in cerebral cortex of 30-day-old Wistar rats in vitro. CO2 production was significantly inhibited (30-70%) by 2-hydroxybutyric acid in cerebral cortex prisms, in total homogenates and in the mitochondrial fraction. We also demonstrated a significant inhibition of lipid synthesis (20-45%) in cerebral cortex prisms and total homogenates in the presence of 2-hydroxybutyric acid. However, no inhibition of lipid synthesis occurred in homogenates free of nuclei and mitochondria. The results indicate an impairment of mitochondrial energy metabolism caused by 2-hydroxybutyric acid, a fact that may secondarily lead to reduction of lipid synthesis. It is possible that these findings may be associated with the neuropathophysiology of the situations where 2-hydroxybutyric acid is accumulated.

Determination of serum aluminum, platelet aggregation and lipid peroxidation in hemodialyzed patients

Aluminum (Al3+) overload is frequently associated with lipid peroxidation and neurological disorders. Aluminum accumulation is also reported to be related to renal impairment, anemia and other clinical complications in hemodialysis patients. The aim of the present study was to determine the degree of lipid peroxidation, platelet aggregation and serum aluminum in patients receiving regular hemodialytic treatment. The level of plasma lipid peroxidation was evaluated on the basis of thiobarbituric acid reactive substances (TBARS). Mean platelet peroxidation in patients undergoing hemodialysis was significantly higher than in normal controls (2.7 ± 0.03 vs 1.8 ± 0.06 nmol/l, P<0.05). Platelet aggregation and serum aluminum levels were determined by a turbidimetric method and atomic absorption spectrophotometry, respectively. Serum aluminum was significantly higher in patients than in normal controls (44.5 ± 29 vs 10.8 ± 2.5 µg/l, P<0.05). Human blood platelets were stimulated with collagen (2.2 µg/ml), adenosine diphosphate (6 µM) and epinephrine (6 µM) and showed reduced function with the three agonists utilized. No correlation between aluminum levels and platelet aggregation or between aluminum and peroxidation was observed in hemodialyzed patients.

DNA-lipid systems: A physical chemistry study

It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection.

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

Reconstitution of membrane proteins into lipid bilayers is a powerful tool to analyze functional as well as structural areas of membrane protein research. First, the proper incorporation of a purified membrane protein into closed lipid vesicles, to produce proteoliposomes, allows the investigation of transport and/or catalytic properties of any membrane protein without interference by other membrane components. Second, the incorporation of a large amount of membrane proteins into lipid bilayers to grow crystals confined to two dimensions has recently opened a new way to solve their structure at high resolution using electron crystallography. However, reconstitution of membrane proteins into functional proteoliposomes or 2-D crystallization has been an empirical domain, which has been viewed for a long time more like "black magic" than science. Nevertheless, in the last ten years, important progress has been made in acquiring knowledge of lipid-protein-detergent interactions and has permitted to build upon a set of basic principles that has limited the empirical approach of reconstitution experiments. Reconstitution strategies have been improved and new strategies have been developed, facilitating the success rate of proteoliposome formation and 2-D crystallization. This review deals with the various strategies available to obtain proteoliposomes and 2-D crystals from detergent-solubilized proteins. It gives an overview of the methods that have been applied, which may be of help for reconstituting more proteins into lipid bilayers in a form suitable for functional studies at the molecular level and for high-resolution structural analysis.

Skin secretion of Siphonops paulensis (Gymnophiona, Amphibia) forms voltage-dependent ionic channels in lipid membranes

The effect of the skin secretion of the amphibian Siphonops paulensis was investigated by monitoring the changes in conductance of an artificial planar lipid bilayer. Skin secretion was obtained by exposure of the animals to ether-saturated air, and then rinsing the animals with distilled water. Artificial lipid bilayers were obtained by spreading a solution of azolectin over an aperture of a Delrin cup inserted into a cut-away polyvinyl chloride block. In 9 of 12 experiments, the addition of the skin secretion to lipid bilayers displayed voltage-dependent channels with average unitary conductance of 258 ± 41.67 pS, rather than nonspecific changes in bilayer conductance. These channels were not sensitive to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid or tetraethylammonium ion, but the experimental protocol used does not permit us to specify their characteristics.

Arachidonic acid triggers an oxidative burst in leukocytes

The change in cellular reducing potential, most likely reflecting an oxidative burst, was investigated in arachidonic acid- (AA) stimulated leukocytes. The cells studied included the human leukemia cell lines HL-60 (undifferentiated and differentiated into macrophage-like and polymorphonuclear-like cells), Jurkat and Raji, and thymocytes and macrophages from rat primary cultures. The oxidative burst was assessed by nitroblue tetrazolium reduction. AA increased the oxidative burst until an optimum AA concentration was reached and the burst decreased thereafter. In the leukemia cell lines, optimum concentration ranged from 200 to 400 µM (up to 16-fold), whereas in rat cells it varied from 10 to 20 µM. Initial rates of superoxide generation were high, decreasing steadily and ceasing about 2 h post-treatment. The continuous presence of AA was not needed to stimulate superoxide generation. It seems that the NADPH oxidase system participates in AA-stimulated superoxide production in these cells since the oxidative burst was stimulated by NADPH and inhibited by N-ethylmaleimide, diphenyleneiodonium and superoxide dismutase. Some of the effects of AA on the oxidative burst may be due to its detergent action. There apparently was no contribution of other superoxide-generating systems such as xanthine-xanthine oxidase, cytochromes P-450 and mitochondrial electron transport chain, as assessed by the use of inhibitors. Eicosanoids and nitric oxide also do not seem to interfere with the AA-stimulated oxidative burst since there was no systematic effect of cyclooxygenase, lipoxygenase or nitric oxide synthase inhibitors, but lipid peroxides may play a role, as indicated by the inhibition of nitroblue tetrazolium reduction promoted by tocopherol.

Lipid peroxidation and total radical-trapping potential of the lungs of rats submitted to chronic and sub-chronic stress

Exposure to stress induces a cluster of physiological and behavioral changes in an effort to maintain the homeostasis of the organism. Long-term exposure to stress, however, has detrimental effects on several cell functions such as the impairment of antioxidant defenses leading to oxidative damage. Oxidative stress is a central feature of many diseases. The lungs are particularly susceptible to lesions by free radicals and pulmonary antioxidant defenses are extensively distributed and include both enzymatic and non-enzymatic systems. The aim of the present study was to determine lipid peroxidation and total radical-trapping potential (TRAP) changes in lungs of rats submitted to different models of chronic stress. Adult male Wistar rats weighing 180-230 g were submitted to different stressors (variable stress, N = 7) or repeated restraint stress for 15 (N = 10) or 40 days (N = 6) and compared to control groups (N = 10 each). Lipid peroxidation levels were assessed by thiobarbituric acid reactive substances (TBARS), and TRAP was measured by the decrease in luminescence using the 2-2'-azo-bis(2-amidinopropane)-luminol system. Chronic variable stress induced a 51% increase in oxidative stress in lungs (control group: 0.037 ± 0.002; variable stress: 0.056 ± 0.007, P < 0.01). No difference in TBARS was observed after chronic restraint stress, but a significant 57% increase in TRAP was presented by the group repeatedly restrained for 15 days (control group: 2.48 ± 0.42; stressed: 3.65 ± 0.16, P < 0.05). We conclude that different stressors induce different effects on the oxidative status of the organism.

Effect of dietary fat saturation on lipid metabolism, arachidonic acid turnover and peritoneal macrophage oxidative stress in mice

We investigated the effects of a saturated fat diet on lipid metabolism and arachidonic acid (AA) turnover in mouse resident peritoneal macrophages. The pro-oxidative effect of this diet was also studied. Female C57BL/6 mice were weaned at 21 days of age and assigned to either the experimental diet containing coconut oil (COCO diet), or the control diet containing soybean oil as fat source (10 mice per group). The fat content of each diet was 15% (w/w). Mice were fed for 6 weeks and then sacrificed. The concentration of total lipids, triglycerides, (LDL + VLDL)-cholesterol, thiobarbituric acid-reactive substances (TBARS) and reduced glutathione were increased in the plasma of mice fed the COCO diet, without changes in phospholipid or total cholesterol concentrations compared to control. The concentrations of total cholesterol, free and esterified cholesterol, triglycerides, and TBARS were increased in the macrophages of COCO-fed mice, while the content of total phospholipids did not change. The phospholipid composition showed an increase of phosphatidylcholine and a decrease of phosphatidylethanolamine. The [³H]-AA distribution in the phospholipid classes showed an increase in phosphatidylcholine and phosphatidylethanolamine. Incorporation of [³H]-cholesterol into the macrophages of COCO-fed mice and into the cholesterol ester fraction was increased. The COCO diet did not affect [³H]-AA uptake but induced an increase in [³H]-AA release. The COCO diet also enhanced AA mobilization induced by lipopolysaccharide. These results indicate that the COCO diet, high in saturated fatty acids, alters the lipid metabolism and AA turnover of peritoneal macrophages in female mice and also produces a significant degree of oxidative stress.

Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 µmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10%) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

Mitochondrial K+ transport and cardiac protection during ischemia/reperfusion

Mitochondrial ion transport, oxidative phosphorylation, redox balance, and physical integrity are key factors in tissue survival following potentially damaging conditions such as ischemia/reperfusion. Recent research has demonstrated that pharmacologically activated inner mitochondrial membrane ATP-sensitive K+ channels (mitoK ATP) are strongly cardioprotective under these conditions. Furthermore, mitoK ATP are physiologically activated during ischemic preconditioning, a procedure which protects against ischemic damage. In this review, we discuss mechanisms by which mitoK ATP may be activated during preconditioning and the mitochondrial and cellular consequences of this activation, focusing on end-effects which may promote ischemic protection. These effects include decreased loss of tissue ATP through reverse activity of ATP synthase due to increased mitochondrial matrix volumes and lower transport of adenine nucleotides into the matrix. MitoK ATP also decreases the release of mitochondrial reactive oxygen species by promoting mild uncoupling in concert with K+/H+ exchange. Finally, mitoK ATP activity may inhibit mitochondrial Ca2+ uptake during ischemia, which, together with decreased reactive oxygen release, can prevent mitochondrial permeability transition, loss of organelle function, and loss of physical integrity. We discuss how mitochondrial redox status, K+ transport, Ca2+ transport, and permeability transitions are interrelated during ischemia/reperfusion and are determinant factors regarding the extent of tissue damage.

APOA1/C3/A4 gene cluster variability and lipid levels in Brazilian children

Genetic studies have suggested that polymorphisms of genes coding for apolipoproteins are significant determinants of serum lipoprotein and lipid levels in adults. However, only a few studies have investigated the association of these polymorphisms in children. Therefore, in the present investigation we studied the distribution of APOA1 -75 G>A, +83 C>T, APOC3 -482 C>T, -455 T>C and 3238 C>G, and APOA4 Q360H and T347S polymorphisms and their influence on plasma lipoprotein levels in children from a Brazilian northeastern admixed population. The seven polymorphic sites were genotyped in 414 children aged 5 to 15 years (mean 8.9 ± 2.9). The genotypes of the seven polymorphic sites were assessed by PCR-RFLP methods. The frequencies of the less common alleles were, in general, intermediate among parental populations, as expected. Strong linkage disequilibrium was detected between polymorphisms at the APOA1, APOC3 and APOA4 loci in this admixed population sample. Overall the genotype effects seen in adults were weaker or absent in children. The APOC3/-455 and APOA4 T347S variants showed significant effects on HDL cholesterol in girls (P = 0.033 and P = 0.016, respectively). Significantly higher plasma total (P = 0.003) and LDL cholesterol (P = 0.004) levels were observed in boys who were carriers of the 3238G allele at the APOC3/3238 C>G site. These results disclosed an overall absence of associations between these polymorphisms and lipids in children. This finding is not unexpected because expression of the effect of these polymorphisms might depend on the interaction with environmental variables both internal and external to the individual.

Atherogenic lipid profile of Brazilian near-term newborns

Cardiovascular disease is the primary cause of death in Brazil. Recent studies have shown that low birth weight and preterm birth are linked to a higher prevalence of cardiovascular disease. The aim of the present study was to compare the levels of lipids and apolipoproteins and atherogenic indexes between term and near-term newborn infants. A sample of umbilical cord blood was obtained from 135 newborns (66 males) divided into two groups: 25 near-term neonates (35-36.6 weeks of gestational age) and 110 term neonates (37-42 weeks of gestational age). The total cholesterol concentrations were higher in the near-term neonates than in the term group (94.04 ± 8.02 vs 70.42 ± 1.63 mg/dl, P < 0.01), due to an increase in the LDL-cholesterol fraction in the near-term group (57.76 ± 6.39 vs 34.38 ± 1.29 mg/dl, P < 0.001). The atherogenic indexes (total cholesterol/HDL-cholesterol, LDL-cholesterol/HDL-cholesterol and apolipoprotein B/apolipoprotein A-I) were higher in the near-term group (P < 0.001, P < 0.001, and P < 0.05, respectively). The gestational age of the newborns was inversely correlated with total cholesterol and LDL-cholesterol, and also with the total cholesterol/HDL-cholesterol and LDL-cholesterol/HDL-cholesterol indexes. These findings demonstrate that the lipid profile is worse in the group of near-term neonates compared with the term group. Future studies are needed to determine if this atherogenic profile in near-term neonates can affect body metabolism, increasing the risk for cardiovascular diseases in adult life.

Towards sustainable transport: A comparison of demographic and behavioural characteristics of Finnish and international car sharing users

SStrong evidence suggests that the climate is changing and that these changes are largely caused by human activities. A consensus exists among researchers that human activity is causing global warming and that actions to mitigate global warming need to be taken swiftly. The transportation sector, which relies heavily on fossil fuel burning and primarily oil, is one of the big contributors to air pollution problems at local, regional and global levels. It is the fastest growing source of greenhouse gas emissions and is estimated to be responsible for nearly a quarter of global energyrelated carbon dioxide emissions. Car sharing is a mobility solution encouraging its users to decrease private car usage in favour of communal transit and environmental goals. The idea of car sharing originates from the aspiration to decrease personal car ownership and to reduce vehicle distance travelled. This thesis seeks to complement the understanding of Finnish car sharing users and their usage through better categorization. Through better categorization and segmentation of Finnish car sharing users the thesis seeks to provide information for improved marketing insight. Research is done on the demographic and behavioural characteristics of Finnish car sharing users and they are compared with international findings about the characteristics of International car sharing users. The main research problem is Are Finnish car sharing users similar to international ones? A theoretical research framework on the determinants of individual car sharing usage is built based on international research about demographic and behaviouristic characteristics. After this a quantitative survey is performed to the customers of a Finnish car sharing organization. The data analysed in the thesis consist out of 532 answers received from the car sharing organizations customers. The data is analysed with descriptive and other exploratory methods, which create an understanding of Finnish car sharing users. At the end of the analysis the demographic and behavioural characteristics of Finnish car sharing users are compared with international ones. The research findings of the thesis indicate that the demographic and behavioural characteristics of Finnish car sharing usage largely follow those of their international counterparts. Thanks to the thesis results the car sharing organization is able to better target their customers through improved marketing insight.