Effect of Brazil nut supplementation on the blood levels of selenium and glutathione peroxidase in hemodialysis patients

Objective: In patients who have undergone hemodialysis, large amounts of reactive oxygen species (ROS) are produced and, at higher concentrations, ROS are thought to be involved in the pathogenesis of cardiovascular disease. It has been proposed that selenium (Se) may exert an anti-atherogenic influence by reducing oxidative stress. The richest known food source of selenium is the Brazil nut (Bertholletia excelsa, family Lecythidaceae), found in the Amazon region. We evaluated the effect of Brazil nut supplementation on blood levels of Se and glutathione peroxidase (GSH-Px) activity in patients on hemodialysis. Methods: A total of 81 patients on hemodialysis (52.0 +/- 15.2 y old, average time on dialysis 82.3 +/- 91.4 mo, body mass index 24.9 +/- 4.4 kg/m(2)) from the RenalCor and RenalVida Clinics in Rio de Janeiro, Brazil, were studied. All patients received one nut (around 5 g, averaging 58.1 mu g Se/g) a day for 3 mo. The Se concentrations in the nuts and in plasma and erythrocytes were determined by atomic absorption spectrophotometry with hydride generation (Hitachi, Z-500). GSH-Px levels were measured using Randox commercial kits. Results: Plasma Se (18.8 +/- 17.4 mu g/L) and erythrocyte (72.4 +/- 37.9 mg/L) levels were below the normal, range before nut supplementation. After supplementation, the plasma level increased to 104.0 +/- 65.0 mu g/L and erythrocytes to 244.1 +/- 119.5 mg/L (P<0.0001). The activity of GSH-Px also increased after supplementation, from 46.6 +/- 14.9 to 55.9 +/- 23.6 U/g of hemoglobin (P<0.0001). Before supplementation, 11% of patients had GSH-Px activity below the normal range (27.5-73.6 U/g of hemoglobin). After supplementation, all patients showed GSH-Px activity within the normal range. Conclusion: The data revealed that the investigated patients presented Se deficiency and that the consumption of only one Brazil nut a day (5 g) during 3 mo was effective to increase the Se concentration and GSH-Px activity in these patients, thus improving their antioxidant status. (C) 2010 Elsevier Inc. All rights reserved.

Associations between glutathione peroxidase-1 Pro198Leu polymorphism, selenium status, and DNA damage levels in obese women after consumption of Brazil nuts

Objective: Alterations in selenium (Se) status may result in suboptimal amounts of selenoproteins, which have been associated with increased oxidative stress levels. The Pro198Leu polymorphism at the glutathione peroxidase-1 (GPx1) gene is supposed to be functional. The response of Se status, GPx activity, and levels of DNA damage to a Se supplementation trial between the genotypes related to that polymorphism was investigated. Methods: A randomized trial was conducted with 37 morbidly obese women. Participants consumed one Brazil nut, which provided approximately 290 mu g of Se a day, for 8 wk. Blood Se concentrations, erythrocyte GPx activity, and DNA damage levels were measured at baseline and at 8 wk. The results were compared by genotypes. Results: The genotype frequencies were 0.487, 0.378, and 0.135 for Pro/Pro (the wild-type genotype), Pro/Leu, and Leu/Leu, respectively. At baseline, 100% of the subjects were Se deficient, and after the supplementation, there was an improvement in plasma Se (P < 0.001 for Pro/Pro and Pro/Leu, P < 0.05 for Leu/Leu), erythrocyte Se (P = 0.00 for Pro/Pro and Pro/Leu, P < 0.05 for Leu/Leu), and GPx activity (P = 0.00 for Pro/Pro, P < 0.00001 for Pro/Leu, P < 0.001 for Leu/Leu). In addition, the Pro/Pro group showed a decrease in DNA damage after Brazil nut consumption compared with baseline (P < 0.005), and those levels were higher in Leu/Leu subjects compared with those with the wild-type genotype (P < 0.05). Conclusion: Consumption of one unit of Brazil nuts daily effectively increases Se status and increases GPx activity in obese women, regardless of GPx1 Pro198Leu polymorphism. However, the evaluated biomarkers showed distinct results in response to the supplementation when the polymorphism was considered. (c) 2011 Elsevier Inc. All rights reserved.

Effects of oral supplementation with glutamine and alanyl-glutamine on glutamine, glutamate, and glutathione status in trained rats and subjected to long-duration exercise

Objective: We investigated the effect of supplementation with the dipeptide L-alanyl-L-glutamine (DIP) and a solution containing L-glutamine and L-alanine, both in the free form, on the plasma and tissue concentrations of glutamine, glutamate, and glutathione (GSH) in rats subjected to long-duration exercise. Methods: Rats were subjected to sessions of swim training. Twenty-one days before sacrifice, the animals were supplemented with DIP (1.5 g/kg, n = 6), a solution of free L-glutamine (1 g/kg) and free L-alanine (0.61 g/kg; GLN + ALA, n = 6), or water (CON, n = 6). Animals were sacrificed before (TR, n = 6) or after (LD, n = 6) long-duration exercise. Plasma concentrations of glutamine, glutamate, glucose, and ammonia and liver and muscle concentrations of glutamine, glutamate, and reduced and oxidized (GSSG) GSH were measured. Results: Higher concentrations of plasma glutamine were found in the DIP-TR and GLN + ALA-TR groups. The CON-LD group showed hyperammonemia, whereas the DIP-LD and GLN + ALA-LD groups exhibited lower concentrations of ammonia. Higher concentrations of glutamine, glutamate, and GSH/GSSG in the soleus muscle and GSH and GSH/GSSG in the liver were observed in the DIP-TR and GLN + ALA-TR groups. The DIP-LD and GLN + ALA-LD groups exhibited higher concentrations of GSH and GSH/GSSG in the soleus muscle and liver compared with the CON-LD group. Conclusion: Chronic oral administration of DIP and free GLN + ALA before long-duration exercise represents an effective source of glutamine and glutamate, which may increase muscle and liver stores of GSH and improve the redox state of the cell. (C) 2009 Published by Elsevier Inc.

Role of promoter polymorphisms in the plasma glutathione peroxidase (GPx-3) gene as a risk factor for cerebral venous thrombosis

Background and Purpose-Plasma glutathione peroxidase (GPx-3) is a major antioxidant enzyme in plasma and the extracellular space that scavenges reactive oxygen species produced during normal metabolism or after oxidative insult. A deficiency of this enzyme increases extracellular oxidant stress, promotes platelet activation, and may promote oxidative posttranslational modification of fibrinogen. We recently identified a haplotype (H-2) in the GPx-3 gene promoter that increases the risk of arterial ischemic stroke among children and young adults. Methods-The aim of this study is to identify possible relationships between promoter haplotypes in the GPx-3 gene and cerebral venous thrombosis (CVT). We studied the GPx-3 gene promoter from 23 patients with CVT and 123 young controls (18 to 45 years) by single-stranded conformational polymorphism and sequencing analysis. Results-Over half of CVT patients (52.1%) were heterozygous (H1H2) or homozygous (H2H2) carriers of the H-2 haplotype compared with 12.2% of controls, yielding a more than 10-fold independent increase in the risk of CVT (OR=10.7; 95% CI, 2.70 to 42.36; P<0.0001). Among women, the interaction of the H2 haplotype with hormonal risk factors increased the OR of CVT to almost 70 (P<0.0001). Conclusions-These findings show that a novel GPx-3 promoter haplotype is a strong, independent risk factor for CVT. As we have previously shown that this haplotype is associated with a reduction in transcriptional activity, which compromises antioxidant activity and antithrombotic benefits of the enzyme, these results suggest that a deficiency of GPx-3 leads to a cerebral venous thrombophilic state.

The Protective Effect of CAPE on Hepatic Ischemia/Reperfusion Injury in Rats

Background/Aims. The transcription factor nuclear factor-kappa B (NF-kappa B) exerts a pivotal role in the pathogenesis of hepatic ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent and specific NF-kappa B inhibitor, presents protective effects on I/R injury in some tissues. This study aimed to evaluate the effect of CAPE on hepatic I/R injury in rats. Materials and methods. Wistar rats were submitted to a sham operation, 60 min ischemia, or 60 min ischemia plus saline or CAPE treatment followed by 6 h reperfusion. Liver tissue injury was evaluated by alanine aminotransferase, aspartate aminotransferase, and tissue glutathione measurement, and histological damage score. Apoptotic hepatocytes were determined by the transferase-mediated dUTP-biotin nick-end labeling assay. Hepatic neutrophil accumulation was assessed by the naphthol method. Lipid peroxidation and NF-kappa B activation were evaluated by 4-hydroxynonenal and NF-kappa B p65 immunohistochemistry, respectively. Results. Animals submitted to ischemia showed a marked increase of alanine aminotransferase and aspartate aminotransferase after reperfusion, but with lower levels in CAPE group. Tissue glutathione content declined gradually during ischemia to reperfusion and was partially recovered with CAPE treatment. The histological damage score, apoptosis index, and neutrophil infiltration, as well as 4-hydroxynonenal and NF-kappa B p65 nuclear labeling, were higher in the liver of animals submitted to I/R compared to the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. Conclusions. CAPE was able to protect the liver against normothermic I/R injury in rats. This effect may be associated with the inhibition of the NF-kappa B signaling pathway and decrease of the acute inflammatory response following I/R in the liver. (C) 2008 Elsevier Inc. All rights reserved.

Effect of NF kappa B Inhibition by CAPE on Skeletal Muscle Ischemia-Reperfusion Injury

Background/Aims. Nuclear factor kappa B (NF kappa B) plays important role in the pathogenesis of skeletal muscle ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent NF kappa B inhibitor, exhibits protective effects on I/R injury in some tissues. In this report, the effect of CAPE on skeletal muscle I/R injury in rats was studied. Methods. Wistar rats were submitted to sham operation, 120-min hindlimb ischemia, or 120-min hindlimb ischemia plus saline or CAPE treatment followed by 4-h reperfusion. Gastrocnemius muscle injury was evaluated by serum aminotransferase levels, muscle edema, tissue glutathione and malondialdehyde measurement, and scoring of histological damage. Apoptotic nuclei were determined by a terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay. Muscle neutrophil and mast cell accumulation were also assessed. Lipoperoxidation products and NF kappa B were evaluated by 4-hydroxynonenal and NF kappa B p65 immunohistochemistry, respectively. Results. Animals submitted to ischemia showed a marked increase in aminotransferases after reperfusion, but with lower levels in the CAPE group. Tissue glutathione levels declined gradually during ischemia to reperfusion, and were partially recovered with CAPE treatment. The histological damage score, muscle edema percentage, tissue malondialdehyde content, apoptosis index, and neutrophil and mast cell infiltration, as well as 4-hydroxynonenal and NF kappa B p65 labeling, were higher in animals submitted to I/R compared with the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. Conclusions. CAPE was able to protect skeletal muscle against I/R, injury in rats. This effect may be associated with the inhibition of the NF kappa B signaling pathway and decrease of the tissue inflammatory response following skeletal muscle I/R. (C) 2009 Elsevier Inc. All rights reserved.

The influence of glutathione modulators on the course of Leishmania major infection in susceptible and resistant mice

Glutathione (GSH) has an important dual role in parasite-host relationship in Leishmania major infection. Our previous studies showed that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in the protection of Leishmania against the toxic effect of nitrogen-derived reactive species. On the other hand, GSH also is very important to the modulation of the effective immune response, inducting NO production and leishmanicidal activity of macrophages. In the present study, we investigated the role of host GSH during the course of L. major infection, analysing the size of footpad lesions and parasite load from mice treated with two GSH modulators, N-acethyl-L-cysteine (NAC) and buthionine sulphoximine (BSO). Resistant mice treated with BSO, which depletes GSH develop exacerbated lesions, but only harbour higher parasite load in their lesions 2 weeks post-infection. Although the NAC treatment does not affect the footpad lesions development in susceptible BALB/c mice, it significantly reduced the tissue parasitism in the lesions throughout the course of infection. Interestingly, the treatment with BSO did not change the course of L. major infection on susceptible mice when compared with nontreated mice. These results suggest that GSH is an important antioxidant modulator during anti-Leishmania immune response in vivo.

Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: Evaluation of reduced glutathione levels and matrix metalloproteinase secretion

Background and purpose: Calendula officinalis flowers have long been employed time in folk therapy, and more than 35 properties have been attributed to decoctions and tinctures from the flowers. The main uses are as remedies for burns (including sunburns), bruises and cutaneous and internal inflammatory diseases of several origins. The recommended doses are a function both of the type and severity of the condition to be treated and the individual condition of each patient. Therefore, the present study investigated the potential use of Calendula officinalis extract to prevent UV irradiation-induced oxidative stress in skin. Methods: Firstly, the physico-chemical composition of marigold extract(ME) (hydroalcoholic extract)was assessed and the in vitro antioxidant efficacy was determined using different methodologies. Secondly, the cytotoxicity was evaluated in L929 and HepG2 cells with the MTT assay. Finally, the in vivo protective effect of ME against UVB-induced oxidative stress in the skin of hairless mice was evaluated by determining reduced glutathione (GSH) levels and monitoring the secretion/activity of metalloproteinases. Results and conclusions: The polyphenol, flavonoid, rutin and narcissin contents found in ME were 28.6 mg/g, 18.8 mg/g, 1.6 mg/g and 12.2 mg/g, respectively and evaluation of the in vitro antioxidant activity demonstrated a dose-dependent effect of ME against different radicals. Cytoxicity experiments demonstrated that ME was not cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of 15 mg/mL However, concentrations greater than or equal to 30 mg/mL, toxic effects were observed. Finally, oral treatment of hairless mice with 150 and 300 mg/kg of ME maintained GSH levels close to non-irradiated control mice. In addition, this extract affects the activity/secretion of matrix metalloproteinases 2 and 9 (MMP-2 and -9) stimulated by exposure to UVB irradiation. However, additional studies are required to have a complete understanding of the protective effects of ME for skin. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Farnesoic acid O-methyl transferase (FAMeT) isoforms: Conserved traits and gene expression patterns related to caste differentiation in the stingless bee, Melipona scutellaris

Farnesoic acid O-methyl transferase (FAMeT) is the enzyme that catalyzes the formation of methyl farnesoate (MF) from farnesoic acid (FA) in the biosynthetic pathway of juvenile hormone (JH). This work reports the cloning, sequencing, and expression of FAMeT gene from the stingless bee Melipona scutellaris (MsFAMeT). The MsFAMeT in silica analysis showed that greatest sequence similarity is found in Apis mellifera and other insects, while relatively less similarity is shown in crustaceans. Evidence of alternative splicing of a 27 nucleotide (nt) microexon explains the presence of the detected isoforms, 1 and 2. The expression analysis of the two isoforms showed a marked difference when castes were compared, suggesting that they could be involved differently in the JH metabolism in M. scutellaris, providing new insights for the comprehension of female plasticity.

REDUCED ERYTHROCITARY GLUTATHIONE AND HEINZ BODIES IN CATS EXPERIMENTALLY INFECTED WITH THE FELINE IMMUNODEFICIENCY VIRUS

Santos, K.B.; Daniel, A.G.T. & Hagiwara, M.K. [Reduced erythrocitary Glutathione and Heinz bodies in cats experimentally infected with the Feline Immunodeficiency Virus.] Glutationa Reduzida Eritrocitaria e Corpusculos de Heinz em gatos infectados experimentalmente pelo Virus da Imunodeficieneia Felina. Revista Brasileira de Medicina Veterinaria, 30(1):26-30, 2008. Centro Universitario Serra dos Orgaos, Rua Comendador Queiroz 52, Apto. 1403, Niteroi, RJ 24230-220, Brasil. E-mail: keilabsantos@)gmail.com The Feline Immunodeficiency Virus (FIV) produces a chronic infection with immunologic system disfunctions in domestic cats developing non-responsive anaemia. The feline immunologic status depression produces free radicals, whose imbalance between production and removal in the organism favour the oxidative injury occurrence. Heinz bodies in large amounts also can evident in vivo oxidative damage. Glutatione, a tripeptide found in the animal cells, is an important protection mechanism against oxidative damages. In this work erythrocyte reduced glutathione (GSH) concentrations for healthy and cats experimentally infected by FIV were determinated in relation to the hemogram and the Heinz bodies presence. All of the animals presents normal values for hemogram and Heinz bodies, however the GSH erythrocyte concentrations in the FIV positive cats were below the normality probably due to an passive depletion to GSH.

Influence of Ascorbic Acid and Glutathione Antioxidants on Frozen-Thawed Canine Semen

Poor sperm viability post-thaw has resulted in constant research into methods of cryopreservation of canine semen. One factor that may be involved in poor viability is sperm oxidative stress caused by excessive formation of reactive oxygen species. The present study was performed in order to evaluate the effect of different concentrations of ascorbic acid (AA) and glutathione (Glu) added to an extender for the freeze-thawing of dog sperm. Semen from five mature dogs was collected and frozen in two studies. Prior to and after freezing, sperm motility, morphology and membrane status were examined. In addition, sperm motility was examined up to 120 min after thawing to evaluate thermo-resistance. In study I, semen was collected twice from each dog. On both occasions, semen was divided into three aliquots: control, Glu 1 mM and Glu 5 mM. In study II, semen was collected twice and divided into three aliquots; control, AA 50 mu M and AA 250 mu M. Initial sperm motility was significantly higher in sperm diluted with AA 50 mu M; sperm longevity, however, measured by a thermal-resistance test (TRT), was higher for Glu treatments. Higher concentration of Glu produced significant improvement in TRT and membrane status, whereas higher concentration of AA had a negative impact in sperm longevity. Antioxidant supplementation to semen freezing extenders improved semen quality post-thaw. Moreover, Glu had the most beneficial effect when supplemented at 5 mM.

Hepatic function evolution in dogs anesthetized with Zolazepam/Tiletamine

The hepatic effects of the anesthetic association zolazepam/tiletamine were investigated in dogs by analyzing the serum concentration of hepatic enzymes. Ten healthy dogs were divided into two groups of five, group I (GI) and group II (GII). The animals of GI received a single dose of 6,6 mg/kg of zolazepam/tiletamine, by intramuscular (IM) injection. GII dogs received 6,6 mg/kg of zolazepam/tiletamine by the IM route; after a period of 50 - 80 minutes the animals received two additional doses (3,3 mg/kg) by intravenous administration[SAH1]. The hepatic function were analyzed by monitoring the serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl-transferase (GGT). Four blood samples were collected in different moments during the analyses: M0, before the first application of the drug; and M1 to M4. M1 through M3 was collected with intervals of 20 minutes before M0, while M4 was obtained 24 hours after M1. The normality of the obtained results was analyzed by Kolmogorov-Smirnov Test; while the Tukey`s test compared the means, using a level of significance of 5% for both statistical analyses. The mean values of all enzymes evaluated were within normal limits for both experimental groups, without any significant statistical alteration being observed between and within these groups. These results demonstrated that the association of zolazepam/tiletamine at the dosage of 6.6 mg/kg, followed by two applications additional of 3.3 mg/kg resulted in elevation of the evaluated hepatic enzymes without exceeding the physiologic values. Additionally, a single application of 6.6 mg/kg of zolazepam/tiletamine by the intramuscular route resulted in lower values when compared to three applications.

A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation

Objective: Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels. Design: hFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays. Results: LMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in Vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day (p < 0.001) of culturing, decreasing thereafter. Conclusions: Our results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. in this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation. (C) 2009 Elsevier Ltd. All rights reserved.

Tartrate-resistant acid phosphatase activity and glutathione levels are modulated during hFOB 1.19 osteoblastic differentiation

Tartrate-resistant acid phosphatase (TRAP) is a well-known marker of osteoclasts and bone resorption. Here we have investigated whether osteoblast-like cells (hFOB 1.19) present TRAP activity and how would be its pattern of expression during osteoblastic differentiation. We also observed how the osteoblastic differentiation affected the reduced glutathione levels. TRAP activity was measured using the p-nitrophenylphosphate substrate. The osteogenic potential of hFOB 1.19 cells was studied by measuring alkaline phosphatase activity and mineralized nodule formation. Oxidative stress was determined by HPLC and DNTB assays. TRAP activity and the reduced glutathione-dependent microenvironment were modulated during osteoblastic differentiation. During this phase, TRAP activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day, decreasing thereafter. We demonstrate that TRAP activity is modulated during osteoblastic differentiation, possibly in response to the redox state of the cell, since it seemed to depend on suitable levels of reduced glutathione.