Estrogen replacement therapy and cardioprotection: mechanisms and controversies

Epidemiological and case-controlled studies suggest that estrogen replacement therapy might be beneficial in terms of primary prevention of coronary heart disease (CHD). This beneficial effect of estrogens was initially considered to be due to the reduction of low density lipoproteins (LDL) and to increases in high density lipoproteins (HDL). Recent studies have shown that estrogens protect against oxidative stress and decrease LDL oxidation. Estrogens have direct effects on the arterial tissue and modulate vascular reactivity through nitric oxide and prostaglandin synthesis. While many of the effects of estrogen on vascular tissue are believed to be mediated by estrogen receptors alpha and ß, there is evidence for `immediate non-genomic' effects. The role of HDL in interacting with 17ß-estradiol including its esterification and transfer of esterified estrogens to LDL is beginning to be elucidated. Despite the suggested positive effects of estrogens, two recent placebo-controlled clinical trials in women with CHD did not detect any beneficial effects on overall coronary events with estrogen therapy. In fact, there was an increase in CHD events in some women. Mutations in thrombogenic genes (factor V Leiden, prothrombin mutation, etc.) in a subset of women may play a role in this unexpected finding. Thus, the cardioprotective effect of estrogens appears to be more complicated than originally thought and requires more research.

Lipoprotein (a): Structure, Pathophysiology and Clinical Implications

The chemical structure of lipoprotein (a) is similar to that of LDL, from which it differs due to the presence of apolipoprotein (a) bound to apo B100 via one disulfide bridge. Lipoprotein (a) is synthesized in the liver and its plasma concentration, which can be determined by use of monoclonal antibody-based methods, ranges from < 1 mg to > 1,000 mg/dL. Lipoprotein (a) levels over 20-30 mg/dL are associated with a two-fold risk of developing coronary artery disease. Usually, black subjects have higher lipoprotein (a) levels that, differently from Caucasians and Orientals, are not related to coronary artery disease. However, the risk of black subjects must be considered. Sex and age have little influence on lipoprotein (a) levels. Lipoprotein (a) homology with plasminogen might lead to interference with the fibrinolytic cascade, accounting for an atherogenic mechanism of that lipoprotein. Nevertheless, direct deposition of lipoprotein (a) on arterial wall is also a possible mechanism, lipoprotein (a) being more prone to oxidation than LDL. Most prospective studies have confirmed lipoprotein (a) as a predisposing factor to atherosclerosis. Statin treatment does not lower lipoprotein (a) levels, differently from niacin and ezetimibe, which tend to reduce lipoprotein (a), although confirmation of ezetimibe effects is pending. The reduction in lipoprotein (a) concentrations has not been demonstrated to reduce the risk for coronary artery disease. Whenever higher lipoprotein (a) concentrations are found, and in the absence of more effective and well-tolerated drugs, a more strict and vigorous control of the other coronary artery disease risk factors should be sought.

Radiometric studies on the oxidation of (1-14c) fatty acids by drug-susceptible and drug-resistant mycobacteria

A radiometric assay system has been used to study oxidation patterns of (1-14C) fatty acids by drug-susceptible and drug-resistant organisms of the genus Mycobacterium. Two strains of M. tuberculosis susceptible to all drugs, H37Rv and Erdman, were used. Drug-resistant organisms included in this investigation were M. tuberculosis H37Rv resistant to 5 ug/ml isoniazid, M. bovis, M. avium, M. intracellular, M. kansasii and M. chelonei. The organisms were inoculated in sterile reaction vials containing liquid 7H9 medium, 10% ADC enrichment and 1.0 uCi of one of the (1-14C) fatty acids (butyric, hexánoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic). Vials were incubated at 37°C and the 14CO2 envolved was measured daily for 3 days with a Bactec R-301 instrument. Although each individual organism displayed a different pattern of fatty oxidation, these patterns were not distinctive enough for identification of the organism. No combination of fatty acids nor preferential oxidation of long chain or of short chain fatty acids were able to separate susceptible from resistant organisms. Further investigation with a larger number of drug susceptible mycobacteria including assimilation studies and oxidation of other substrates may be required to achieve a distinction between drug-susceptible and drug-resistant mycobacteria.

Radiometric studies on the oxidation of (U-14C) L-amino acids by drug-susceptible and drug-resistant mycobacteria

A radiometric assay system has been used to study oxidation patterns of (U-14C) L-amino acids by drug-susceptible and drug-resistant mycobacteria. Drug-susceptible M. tuberculosis (H37Rv TMC 102 and Erdman) along with the drug-resistant organism M. tuberculosis (H37 Rv TMC 303), M. bovis, M. avium, M. intracellulare, M. kansasii and M. chelonei were used. The organisms were inoculated into a sterile reaction system with liquid 7H9 medium and one of the (U-14C) L-amino acids. Each organism displayed a different pattern of amino acid oxidation, but these patterns were not distinctive enough for identification of the organism. Complex amino acids such as proline, phenylalanine and tyrosine were of no use in identification of mycobacteria, since virtually all organisms failed to oxidize them. There was no combination of substrates able to separate susceptible from resistant organisms.

Alterations in lipid transfer to High-Density Lipoprotein (HDL) and activity of paraoxonase-1 in HIV+ patients

HIV+ patients often develop alterations of the plasma lipids that may implicate in development of premature coronary artery disease. High-density lipoprotein (HDL) has an important role in preventing atherogenesis and the aim of this study was to investigate aspects of HDL function in HIV+ patients. HIV+ patients (n = 48) and healthy control subjects (n = 45) of both sexes with similar age were studied. Twenty-five were not being treated with antiretroviral agents, 13 were under reverse transcriptase inhibitor nucleosidic and non-nucleosidic (NRTI+NNRTI) and 10 were under NRTI + protease inhibitors (NRTI+PI) treatment. Paraoxonase 1 (PON1) activity and the transfer of free and esterified cholesterol, tryglicerides and phospholipids from a lipidic nanoemulsion to HDL were analyzed. In comparison with healthy controls, HIV+ patients presented low PON-1 activity and diminished transfer of free cholesterol and tryglicerides. In contrast, phospholipid transfer was increased in those patients, whereas the transfer of cholesteryl esters was unchanged. NRTI+NNRTI increases the transfer of cholesteryl esters and triglycerides but in NRTI+PI there was no difference in respect to non-treated HIV+ patients. HDL from HIV+ patients has smaller antioxidant properties, as shown by lower PON-1 activity, and the transfer of lipids to this lipoprotein fraction is also altered, suggesting that HDL function is defective in those patients.

Changes in the profile of lipoprotein subfractions associated with hormone replacement therapy

OBJECTIVE: To report the effects of 2 regimens of hormone replacement therapy during the postmenopausal period on the profile of the major lipoprotein subfractions (HDL, LDL, and VLDL). METHODS: We carried out a cohort study in 38 postmenopausal patients who were starting their hormone replacement therapy due to gynecological indications, for a period of 12 weeks. Analysis of lipoprotein subclasses was performed through nuclear magnetic resonance spectroscopy. RESULTS: Hormone replacement therapy cause an increase in the proportion of larger subfractions of VLDL and HDL (p=0.008 and 0.03, respectively) and in the proportion of larger particles of VLDL due to a 36% increase in the levels of larger particles (p=0.004), concomitantly with a 15% reduction in the levels of smaller particles (p=0.04). In regard to HDL, the increase occurred only a 17% increase in the levels of larger particles (p=0.002). No significant change occurred in the distribution pattern of LDL subfractions. CONCLUSION: The proportion of larger subfractions of VLDL and HDL increases after hormone replacement therapy. The increase in the proportion of larger particles of VLDL occurs due to an increase in the levels of the larger subclasses concomitantly with a reduction in the smaller particles. However, an increase in the proportion of larger particles of HDL occurs only due to an increase in the levels of the larger subfractions.

Comparison of the effect of two HMG CoA reductase inhibitors on LDL susceptibility to oxidation

OBJECTIVE: To study the differences between fluvastatin and pravastatin regarding LDL susceptibility to oxidation, plasma levels of total cholesterol (TC), HDL-C, LDL-C and triglycerides (TG) in hypercholesterolemic patients with established coronary heart disease (CHD). METHODS: A double-blind randomized parallel study was conducted that included 41 hypercholesterolemic outpatients with CHD treated at the Instituto de Cardiologia do Rio Grande do Sul. The inclusion criteria were LDL-C above 100 mg/dL and triglycerides below 400 mg/dL based on 2 measures. After 4 weeks on a low cholesterol diet, those patients that fullfilled the inclusion criteria were randomized into 2 groups: the fluvastatin group (fluvastatin 40 mg/day) and the pravastatin group (pravastatin 20 mg/day), for 24 weeks of treatment. LDL susceptibility to oxidation was analyzed with copper-induced production of conjugated dienes (Cu2+) and water-soluble free radical initiator azo-bis (2'-2'amidinopropanil) HCl (AAPH). Spectroscopy nuclear magnetic resonance was used for determination of lipids. RESULTS: After 24 weeks of drug therapy, fluvastatin and pravastatin significantly reduced LDL susceptibility to oxidation as demonstrated by the reduced rate of oxidation (azo and Cu) and by prolonged azo-induced lag time (azo lag). The TC, LDL-C, and TG reduced significantly and HDL-C increased significantly. No differences between the drugs were observed. CONCLUSION: In hypercholesterolemic patients with CHD, both fluvastatin and pravastatin reduced LDL susceptibility to oxidation.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.

Organic sulfur oxidation to sulfate in soil samples for total sulfur determination by turbidimetry

Sulfur in the soil occurs in two basic forms, organic and inorganic S. The organic form accounts for 95 % of S in most soils. The effectiveness of organic S to oxidate to sulfate was evaluated for total S determination in soil samples by wet (acid) and dry-ash (alkaline) oxidation methods. To evaluate the wet method and the possible use as a reference when evaluating the dry method proposed here, a reference standard from the US National Institute of Standards and Technology (NIST) was used (Montana Soil - NIST 2710). The dry-ash oxidation process with alkaline oxidizing agents is one of the simplest oxidation methods of organic S to the sulfate form and was compared with the wet process. The objective of the study was to develop a dry method that would be easy to apply and allow the complete conversion of organic S to sulfate in soil samples and later detection by turbidimetry. The effectiveness of organic S oxidation to sulfate was evaluated by means of three alkaline oxidation mixtures: NaHCO3 + Ag2O, Eschka mixture (17 % Na2CO3, 66 % MgO, and 17 % K2CO3), and NaHCO3 + CuO. The procedure to quantify the sulfate concentration was based on the reaction with barium chloride and turbidimetric detection. Sulfur quantification in the standard sample by the wet method proved adequate, precise and accurate. It should also be pointed out that no significant differences were found (95 % reliability) between the wet and dry processes (NaHCO3 and Ag2O oxidation mixture) in six different Brazilian soils. The proposed dry method can therefore be used in the preparation of soil samples for total S determination.

Nickel incorporation in Fe(II, III) hydroxysulfate Green Rust: effect on crystal lattice spacing and oxidation products

Ni(II)-Fe(II)-Fe(III) layered double hydroxides (LDH) or Ni-containing sulfate green rust (GR2) samples were prepared from Ni(II), Fe(II) and Fe(III) sulfate salts and analyzed with X ray diffraction. Nickel is readily incorporated in the GR2 structure and forms a solid solution between GR2 and a Ni(II)-Fe(III) LDH. There is a correlation between the unit cell a-value and the fraction of Ni(II) incorporated into the Ni(II)-GR2 structure. Since there is strong evidence that the divalent/trivalent cation ratio in GR2 is fixed at 2, it is possible in principle to determine the extent of divalent cation substitution for Fe(II) in GR2 from the unit cell a-value. Oxidation forms a mixture of minerals but the LDH structure is retained if at least 20 % of the divalent cations in the initial solution are Ni(II). It appears that Ni(II) is incorporated in a stable LDH structure. This may be important for two reasons, first for understanding the formation of LDHs, which are anion exchangers, in the natural environment. Secondly, this is important for understanding the fate of transition metals in the environment, particularly in the presence of reduced Fe compounds.

Comparison of the methane oxidation rate in four media

Landfill gas emissions are one of the main sources of anthropogenic methane (CH4), a major greenhouse gas. In this paper, an economically attractive alternative to minimize greenhouse gas emissions from municipal solid waste landfills was sought. This alternative consists in special biofilters as landfill covers with oxidative capacity in the presence of CH4. To improve the quality/cost ratio of the project, compost was chosen as one of the cover substrates and soil (Typic red yellow-silt-clay Podzolic) as the other. The performance of four substrates was studied in laboratory experiments: municipal solid waste (MSW) compost, soil, and two soil-compost at different proportions. This study aimed to evaluate the suitability and environmental compatibility as a means of CH4 oxidation in biofilters. Four biofilters were constructed in 60 cm PVC tubes with an internal diameter of 10 cm. Each filter contained 2.3 L of oxidizing substrate at the beginning of the experiment. The gas used was a mixture of CH4 and air introduced at the bottom of each biofilter, at a flow of 150 mL min-1, by a flow meter. One hundred days after the beginning of the experiment, the best biofilter was the MSW compost with an oxidation rate of 990 g m-3 day-1 , corresponding to an efficiency of 44 %. It can be concluded that the four substrates studied have satisfactory oxidative capacity, and the substrates can be used advantageously as cover substrate of MSW landfills.

Iron oxidation on the surface of adventitious roots and its relation to aerenchyma formation in rice genotypes

Establishment of the water layer in an irrigated rice crop leads to consumption of free oxygen in the soil which enters in a chemical reduction process mediated by anaerobic microorganisms, changing the crop environment. To maintain optimal growth in an environment without O2, rice plants develop pore spaces (aerenchyma) that allow O2 transport from air to the roots. Carrying capacity is determined by the rice genome and it may vary among cultivars. Plants that have higher capacity for formation of aerenchyma should theoretically carry more O2 to the roots. However, part of the O2 that reaches the roots is lost due to permeability of the roots and the O2 gradient created between the soil and roots. The O2 that is lost to the outside medium can react with chemically reduced elements present in the soil; one of them is iron, which reacts with oxygen and forms an iron plaque on the outer root surface. Therefore, evaluation of the iron plaque and of the formation of pore spaces on the root can serve as a parameter to differentiate rice cultivars in regard to the volume of O2 transported via aerenchyma. An experiment was thus carried out in a greenhouse with the aim of comparing aerenchyma and iron plaque formation in 13 rice cultivars grown in flooded soils to their formation under growing conditions similar to a normal field, without free oxygen. The results indicated significant differences in the volume of pore spaces in the roots among cultivars and along the root segment in each cultivar, indicating that under flooded conditions the genetic potential of the plant is crucial in induction of cell death and formation of aerenchyma in response to lack of O2. In addition, the amount of Fe accumulated on the root surface was different among genotypes and along the roots. Thus, we concluded that the rice genotypes exhibit different responses for aerenchyma formation, oxygen release by the roots and iron plaque formation, and that there is a direct relationship between porosity and the amount of iron oxidized on the root surface.

Electrocatalytic oxidation of beta-dicarbonyl compounds using ceric methanesulphonate as mediator

beta-dicarbonyl compounds were oxidized electrocatalytically, with fragmentation and loss of "ch2", using ceric methanesulphonate as a mediator. 2,4-pentanedione yields acetic acid (90%), methyl acetoacetate yields acetic acid (84%) plus methanol and dimethyl malonate yields methanol (64%). For 1,3-diphenyl-1,3-propanedione and 1,3-cyclohexanedione, benzoic acid (61% yield) and glutaric acid (75% yield) were obtained, respectively. Methyl cyanoacetate and malononitrile were inert.