Iron absorption in adults with sickle cell anemia: a stable-isotope approach.

PURPOSE: Iron absorption in sickle cell anemia (SCA) remains unclear and studies in adults with SCA are scarce. The aim of this study was to evaluate the iron absorption SCA adults and its association with iron status and hepcidin concentration. METHODS: SCA patients (n = 13; SCAtotal) and control participants (n = 10) ingested an oral stable iron isotope (57Fe). Iron absorption was measured by inductively coupled plasma mass spectrometry (ICP-MS) 14 days after isotope administration. Patients with ≥ 1000 ng/mL serum ferritin were considered to present iron overload (IO) (SCAio+; n = 3) and others classified without IO (SCAio-; n = 10). RESULTS: Iron absorption in the control group ranged from 0.3 to 26.5% (median = 0.9%), while it varied from 0.3 to 5.4% in SCAio+ (median = 0.5%) and from 0.3 to 64.2% in the SCAio- (median = 6.9%). Hepcidin median values were 14.1 ng/mL (3.0-31.9 ng/mL) in SCAio-, 6.2 ng/mL (3.3-7.8 ng/mL) in SCAio + and 6.2 ng/mL (0.6-9.3 ng/mL) in control. Iron absorption was associated with ferritin level (r = - 0.641; p = 0.018) and liver iron concentration (LIC; r = - 0.786; p = 0.036) in the SCAtotal group. CONCLUSION: Our data suggest that SCAio- individuals may be at risk of developing primary IO. Simultaneously, secondary IO may induce physiological adaptation, resulting in reduced iron absorption. Further studies evaluating intestinal iron absorption using larger sample sizes should be conducted to help establish a safe nutrition approach to be adopted and to ensure the security of food-fortifying public policies for these patients. TRIAL REGISTRATION: This trial was registered at www.ensaiosclinicos.gov.br (Identifier RBR-4b7v8pt).

Distribution of acid-volatile sulfides and simultaneously extracted metals in Guanabara Bay: combination of anthropogenic, sedimentological, and geochemical processes.

Coastal sedimentary systems are affected by continental and marine metal pollutant inputs associated with different hydrodynamic characteristics and geochemical processes. These include the formation of acid-volatile sulfides (AVS) within sediments, which affects metal bioavailability and associated aquatic biota toxicity risks. Physicochemical changes in these environments in the face of extreme natural or man-made environmental influences can dramatically alter metal bioavailability and toxicity through metal binding and immobilization as insoluble sulfides. Surface sediments from Guanabara Bay, river mouths, and two mangrove areas were collected, and AVS and simultaneously extracted metals Cd, Cu, Fe, Mn, Ni, Pb, and Zn and ΣSEM were determined to assess sediment quality. A severe eutrophication history favored AVS concentrations exceeding or close to the sum-SEM concentrations, demonstrating that AVS play an important role in making trace metals unavailable for assimilation by living organisms, mitigating the risks of contamination for the local biota. This eutrophication-driven sulfide accumulation may attenuate the sediment toxicity in sites heavily polluted by metals, while some fewer eutrophic sites became more exposed to metals in excess to AVS.

Novel bioanalytical strategy using isotope pattern deconvolution and ICP-QMS for the study of iron incorporation in erythrocytes: An insight to better assessment.

Iron is an essential element for human life and its nutritional status in the human body is directly linked to human health. More than 1015 atoms of iron per second are necessary for the maintenance of haemoglobin formation. To predict iron bioavailability three approaches are normally employed: (a) faecal recovery; (b) plasma appearance; and (c) erythrocyte incorporation (the most used). Isotope Pattern Deconvolution (IPD) is a mathematical tool that allows the isolation of distinct isotope signatures from mixtures of natural abundance and enriched tracers. In this work we propose a novel strategy to assess erythrocyte iron incorporation, based on the use of an iron stable isotope (57Fe) and the IPD concept. This strategy allows direct calculation of the exogenous concentration of 57Fe incorporated into RBCs after supplementation. In this way, to determine the mass of iron incorporated into erythrocytes, the unique prediction that must be made is the blood volume, estimate to reproduce the natural dilution of the tracer (57Fe) in the blood. This novel bioanalytical approach was applied for the measurements of iron incorporation and further iron absorption studies in humans, using a group of twelve healthy participants, that should be further evaluated for the assessment of other chemical elements that could be of health concerns and directly impact society.

Coordination chemistry applied to anti-doping analysis: Cobalt quantification as its diethyldithiocarbamate complex by liquid chromatography coupled to high resolution tandem mass spectrometry.

Cobalt was included on the World Anti-Doping Agency Prohibited List in 2015 due to its effect on stimulus of erythropoiesis via stabilization of hypoxia-inducible factor. Although it has proven benefits for performance enhancement, the unavailability of inductively coupled plasma-mass spectrometry on routine of the accredited laboratories is a factor that reduces its applicability in anti-doping analysis. Therefore, an analytical method for quantification of urinary cobalt as its diethyldithiocarbamate complex by liquid chromatography coupled with high-resolution tandem mass spectrometry was developed and validated. Palladium was proposed as internal standard and rhodium as a complexation control. A microwave-assisted acid digestion of the urine samples was essential, not only to eliminate the matrix effect but mainly to avoid the non-specific bond of cobalt to endogenous molecules. A linear method was obtained over the studied range from a negative urine control to a spiked concentration of 25 ng/mL, with an estimated limit of quantification of 2.5 ng/mL, and an adequate combined standard uncertainty of 11.4%. Considering that all reagents are commercially available, the proposed strategy is feasible to be included on routine sample preparation. Monitoring urinary cobalt concentrations globally opens the perspective to support the anti-doping system to define a suitable threshold value and to understand its potential misuse by athletes seeking for performance improvement.

Development of an electrochemical sensor based on ternary oxide SiO2/Al2O3/SnO2 modified with carbon black for direct determination of clothianidin in environmental and food samples.

This study presents the development of an electrochemical sensor, denoted as GCE/CB/SiAlSn, based on the modification of a glassy carbon electrode surface with the ternary oxide SiO2/Al2O3/SnO2 associated with carbon black, for direct determination of the neonicotinoid pesticide clothianidin in different matrices, such as environmental and food samples. Morphological characterization by the scanning electron microscopy technique, electroanalytical analyses using the cyclic voltammetry technique and differential pulse voltammetry are presented which demonstrated that the developed electrochemical platform presents high sensitivity in the electroanalytical clothianidin determination. The linear range studied was from 2.99 × 10-7 to 6.04 × 10-5 mol L-1, with an LOD of 2.47 nmol L-1. This high sensitivity was explained using the synergistic relationship between carbon black and ternary oxide that maximized the electroactive surface area of the GCE/CB/SiAlSn sensor. Interferent studies were performed that showed high selectivity of the sensor to the pesticide in the presence of Ca2+, K+, Na+, and Mg2+ and carbendazim, glyphosate, imidacloprid and thiamethoxam pesticides. The sensor was applied to real samples of tap water and apple juice obtaining recoveries from 91.0% to 103.0%.

Evaluation of microplastic contamination by metals in a controlled environment: A risk to be considered.

The metal contamination and the degradation of polyethylene terephthalate (PET) due to human activities have contributed to the worsening of environmental problems in aquatic systems. Therefore, the study aimed to evaluate PET microplastic adsorption levels when exposed to high amounts of Ni, Cu and Co. The PET microplastic was characterized by scanning electron microscopy, Brunner-Emmet-Teller, porosimetry system, Barrett-Joyner-Halenda and Fourier transform infrared spectroscopy with attenuated total reflectance for evaluation of surface morphology, surface area, porosity, pore size and functional groups, respectively. The results showed that the surface area, the presence of macro and mesopores, and the functional groups influence the adsorption of metals on the surface of PET microplastic. The adsorption isotherms confirmed the presence of mesoporosity and macroporosity on the PET microplastic surface. The Freundlich and Langmuir models were used to study the adsorption capacity. The kinetics of adsorptions were interpreted using pseudo-first order and pseudo-second order models. The results indicated that the Langmuir isotherm and the pseudo-second order adequately described the adsorption of metals by the PET microplastic. The removal rates by the PET microplastic varied from 8 to 34% for Ni, 5 to 40% for Cu and 7 to 27% for Co after a period of 5 days. Furthermore, the adsorption was predominantly chemical and extremely fast, indicating that the presence of microplastics in the environment can lead to a rapid metal accumulation which elevates the hazards potential of microplastic in living beings.

Biscuits Prepared with Enzymatically-Processed Soybean Meal Are Rich in Isoflavone Aglycones, Sensorially Well-Accepted and Stable during Storage for Six Months.

Soybean meal (SBM) is a co-product of the soybean oil industry that is rich in bioactive compounds, such as isoflavones. We aimed to study the effects of processing SBM by fermentation (Saccharomyces cerevisiae) (FSBM) and enzymatic hydrolysis (CelluMax C, a commercial cellulase) (ESBM) on its chemical composition, with emphasis on isoflavones. Fermentation increased protein content by 9%, ash content by 7%, dietary fiber by 11% and minerals by up to 38%, except for iron, which decreased by 26%. Fermentation completely removed oligosaccharides from SBM, while enzymatic processing decreased oligosaccharides by 45% in SBM. Both processes converted glycosylated isoflavones into the corresponding aglycones, the content of which increased by up to 7.7-fold. Biscuits containing SBM, FSBM and ESBM could be labeled as dietary sources of dietary fibers, potassium, phosphorous, calcium and zinc, as well as high in proteins, copper, iron, manganese and magnesium. While FSBM biscuits had lower sensory scores compared to SBM biscuits, ESBM biscuits had equivalent scores. During storage for 180 days at room temperature, the isoflavone profile of all biscuits remained stable. Moreover, storage did not impair microbiological and sensory qualities of any biscuits. Altogether, ESBM biscuits show great marketing potential.

An electrochemical sensor-based carbon black associated with a modified mixed oxide (SiO2/TiO2/Sb2O5) for direct determination of thiamethoxam in raw honey and water samples.

The study aimed to develop an electrochemical sensor based on glassy carbon, mixed oxide (SiO2/TiO2/Sb2O5), and carbon black. The material was synthesized, characterized, and used to determine thiamethoxam in raw honey and water. The morphologic structure and electrochemical performance of the sensor was characterized by scanning electron microscopy and cyclic voltammetry. Differential pulse voltammetry with a concentration of 0.1 mol L-1 of Britton-Robinson buffer at pH 7.0 allowed the generation of a method to determine thiamethoxam in a linear range of 0.25 to 100.5 µmol L-1 and with a limit of detection of 0.012 µmol L-1. The system efficiently quantified traces of thiamethoxam in raw honey and tap water samples. The modified sensor did not present interferences of K+, Na+, Ca2+, Mg2+, glyphosate, imidacloprid, and carbendazim. In addition, the device showed good recovery values for thiamethoxam when applied directly to honey and water samples without any treatment, presenting an electrochemical sensor to monitor real-time hazardous substances in environmental and food matrices.

Multivariate optimization of microwave-assisted digestion methods for Cu and Sn determination in antifouling paints using inductively coupled plasma optical emission spectrometry.

Antifouling paints containing Cu, Zn, organotins, and many organic booster biocides may be found in ships and watercraft hulls to avoid the fouling of marine organisms. This type of paint can be harmful to the environment, therefore, the monitoring of toxic elements and compounds in antifouling paints are of great importance to access its quality and potential toxicity to the environment. Hence, this work describes the development of microwave-assisted digestion methods for the determination of Cu and Sn in antifouling paints by inductively coupled plasma optical emission spectrometry (ICP OES). The factors: sample mass and solutions of HNO3, HF, and HCl were optimized using the central composite design (CCD). Dry ashing with a muffle furnace and laser ablation-ICP-MS were used for methodological comparison with the microwave digestion-assisted ICP-OES methods. All the mixtures of acids allowed efficient extraction of the analytes; however, the one that stood out was the use of HF, HNO3, and H2O2.

Ecological risks associated to trace metals of contaminated sediments from a densely urbanized tropical eutrophic estuary.

The sediment contamination by trace metals in coastal aquatic ecosystems is a worldwide environmental problem, since metals can be toxic, persistent, and bioaccumulated. In case of natural events, such as storms, or anthropogenic activities, like dredging, the sediment resuspension to the water column occurs and can solubilize metals, probably increasing their bioavailability and consequently the risk to aquatic life. This study evaluated the bioavailability on reactive trace metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) in estuarine sediments from Iguaçu and Meriti Rivers, both in the drainage basin of Guanabara Bay (Rio de Janeiro, Brazil). Additionally, a discussion about the anthropogenic interference throughout time of six short sediments cores, calculating three different indexes (contamination factors, CF; potential ecological risk index for a single heavy metal, Eif for short; potential ecological risk, PERI) was performed. It was considered as reactive phase, the metal concentrations obtained using a weak acid extraction (in HCl 1 mol L-1 solution). Zn presented high concentrations after resuspension, being above effect range medium (ERM) (52.81 to 1337.4 mg kg-1). The CF indicated very high contamination degree for Cu (14.62 to 17.96) and Zn (27.80 to 35.85) for both rivers. The Eif for short presented higher risk to Cu and Zn for Iguaçu and Meriti rivers. PERI index classified Meriti River samples as severely contaminated (238.10 to 351.62).

Subacute exposure to lead promotes disruption in the thyroid gland function in male and female rats.

Exposure to heavy metals, such as lead, is a global public health problem. Lead has a long historic relation to several adverse health conditions and was recently classified as an endocrine disruptor. The aim of this study was to investigate the effects of subacute exposure to lead on the thyroid gland function. Adult male and female Wistar rats received a lead acetate solution containing 10 or 25 mg/kg, by gavage, three times a week, for 14 days. One week later, behavioral testing showed no alterations in anxiety and motor-exploratory parameters, as evaluated by Open-Field and Plus-Maze Tests, but impairment in learning and memory was found in the male 25 mg/kg lead-treated group and in both female lead-treated groups, as evaluated by the Inhibitory Avoidance Test. After one week, serum levels of tT3 were reduced in the 25 mg/kg female group and in the 10 mg∕ kg male group. However, tT4 levels were increased in the 25 mg/kg male group and in both female treated groups. TSH levels did not change and lead serum levels were undetectable. Morphologic alterations were observed in the thyroid gland, including abnormal thyroid parenchyma follicles of different sizes, epithelial stratification and vacuolization of follicular cells, decrease in colloid eosinophilia and vascular congestion, accompanied by morphometric alterations. An increase in collagen deposition was also observed. No differences were observed in TPO activity or protein expression, H2O2 generation by NADPH oxidases or hepatic D1 mRNA expression. However, thyroid NIS protein expression was considerably decreased in the male and female lead-treated groups, while TSHr expression was decreased in the 25 mg/kg female lead-treated group. These findings demonstrated that subacute exposure to lead acetate disrupts thyroid gland function in both sexes, leading to morphophysiological impairment and to changes in learning and memory abilities.

Total metal content and chemical speciation analysis of iron, copper, zinc and iodine in human breast milk using high-performance liquid chromatography separation and inductively coupled plasma mass spectrometry detection.

The aim of this research was to quantify essential trace elements (iron, copper, zinc and iodine) and establish their speciation in human milk. Both the element and the species are important in new-born nutrition. Colostrum, and transitional and mature milks (25) were collected from 18 mothers of pre-term or full-term infants. Concentrations of the target elements were determined using ICP-MS. For speciation, HPLC coupled to ICP-MS was employed. Total contents of the micronutrients varied in mothers of pre-term (Fe = 0.997, Cu = 0.506, Zn = 4.15 and I = 0.458 mg L-1) and mothers of full-term (Fe = 0.733, Cu = 0.234, Zn = 2.91 and I = 0.255 mg L-1) infants. Fe, Cu and Zn were associated with biomolecules with high molecular mass compounds, such as immunoglobulins, albumin and lactoferrin whilst iodine was only found as iodide.

Água-régia como extrator alternativo para o diagnóstico ambiental de áreas impactadas por resíduos industriais: estudo de caso Centres (Queimados, RJ) / Aqua regia as an alternative extractor for the environmental diagnosis of areas impacted by industrial waste: case study Centres (Queimados, RJ)

RESUMO Áreas contaminadas por metais podem configurar sério risco à saúde pública e ao meio ambiente. Na década de 1980 foi instalado um pátio de estocagem provisória de resíduos industriais perigosos (CENTRES), no município de Queimados, Rio de Janeiro, o que causou um grande impacto ambiental ao solo da região. A legislação brasileira para o diagnóstico de áreas contaminadas limita a extração de metais em solos aos métodos recomendados pela United States Environmental Protection Agency (USEPA) 3050 e 3051A. Entretanto, muitos pesquisadores utilizam o método com água-régia para a extração de metais em solo, mas são escassos estudos que comparem as concentrações de metais extraídos por água-régia e USEPA 3051A. Logo, o objetivo deste trabalho foi avaliar se o método de extração de metais em solos por água-régia é estatisticamente diferente do método USEPA 3051A. Os resultados obtidos revelaram que as concentrações de Cd, Cr, Cu, Ni, Pb e Zn utilizando ambas as extrações (água-régia e USEPA 3051A) foram estatisticamente semelhantes. Também foi possível identificar que as concentrações de Cu, Ni, Pb e Zn foram superiores ao valor de investigação industrial, o que caracteriza essa área como altamente contaminada e solo classe 4.

ABSTRACT Metal contaminated areas lead to environmental and public health risks. In the 1980s, dangerous industrial waste storage was initiated in the CENTRES area in the municipality of Queimados, Rio de Janeiro, Brazil. The inadequate management of these wastes has promoted a large environmental impact to this region's soils. The Brazilian legislation for diagnostics of contaminated areas limits the extraction of metals on soils to USEPA 3050-B and USEPA 3051-A methods. However, many researchers use the aqua regia method for extracting metals in soil, but there are few studies comparing concentrations of metals extracted by aqua regia and USEPA 3051A. Therefore, the objective of this work was to evaluate if the method of extracting metals in soils by aqua regia is statistically different from US EPA 3051A. The results showed that the Cd, Cr, Cu, Ni, Pb and Zn concentrations using both extractions were statistically similar. It was also observed that Cu, Ni, Pb and Zn concentrations were higher than the industrial investigation value which characterizes this area as highly contaminated and soil class 4.

Monitoring bone changes due to calcium, magnesium, and phosphorus loss in rat femurs using Quantitative Ultrasound.

Bone mineral density is an important parameter for the diagnosis of bone diseases, as well as for predicting fractures and treatment monitoring. Thus, the aim of the present study was to evaluate the potential of Quantitative Ultrasound (QUS) to monitor bone changes after calcium, phosphorus, and magnesium loss in rat femurs in vitro during a demineralization process. Four quantitative ultrasound parameters were estimated from bone surface echoes in eight femur diaphysis of rats. The echo signals were acquired during a decalcification process by Ethylenediaminetetraacetic Acid (EDTA). The results were compared to Quantitative Computed Tomography (QCT) and inductively coupled plasma optical emission spectrometry measurements for validation. Integrated Reflection Coefficient (IRC) reflection parameters and Frequency Slope of Reflection Transfer Function (FSRTF) during demineralization tended to decrease, while the backscattering parameter Apparent Integrated Backscatter (AIB) increased and Frequency Slope of Apparent Backscatter (FSAB) showed an oscillatory behavior with no defined trend. Results indicate a clear relation between demineralization and the corresponding decrease in the reflection parameters and increase in the scattering parameters. The trend analysis of the fall curve of the chemical elements showed a better relationship between IRC and QCT. It was possible to monitor bone changes after ions losses, through the QUS. Thus, it is an indication that the proposed protocol has potential to characterize bone tissue in animal models, providing consistent results towards standardization of bone characterization studies by QUS endorsing its use in humans.

Ecological risk evaluation of sediment metals in a tropical Euthrophic Bay, Guanabara Bay, Southeast Atlantic.

Surface sediments were collected from Guanabara Bay, at 14 stations distributed in five sectors, over three sampling campaigns. Analyses of metals, grain size fractions and total organic carbon analyses were performed. The geo-accumulation index and the enrichment factor were estimated to assess contamination status based on background values. Additionally, the sediment quality guidelines were applied to evaluate the adverse biological effects. Results show that there was no seasonal variation in sediment quality based on any methodology, and all methods utilized showed that NW sector and HRJ sector were the worst affected and that the NE sector had the best conditions. The sediments of GB are polluted mainly by Cr, Pb and Zn. According to ΣSEM/AVS, these metals are not available to the biota, although toxicity tests dispute this. Among the various methods employed, those using background values for the area seem to best reflect the local historical contamination.

Total zinc quantification by inductively coupled plasma-mass spectrometry and its speciation by size exclusion chromatography-inductively coupled plasma-mass spectrometry in human milk and commercial formulas: Importance in infant nutrition.

This paper summarises results of zinc content and its speciation in human milk from mothers of preterm and full-term infants at different stages of lactation and from synthetic formula milks. Human milk samples (colostrum, 7th, 14th, and 28th day after delivery) from Spanish and Brazilian mothers of preterm and full-term infants (and also formula milks) were collected. After adequate treatment of the sample, total Zn was determined, while speciation analysis of the Zn was accomplished by size exclusion chromatography coupled online with the ICP-MS. It is observed that total zinc content in human milk decreases continuously during the first month of lactation, both for preterm and full term gestations. All infant formulas analysed for total Zn were within the currently legislated levels. For Zn speciation analysis, there were no differences between preterm and full term human milk samples. Moreover Zn species elute mainly associated with immunoglobulins and citrate in human milk whey. Interestingly the speciation in formula milk whey turned out to be completely different as the observed Zn(2+) was bound almost exclusively to low molecular weight ligands (citrate) and only comparatively very low amounts of the metal appeared to be associated with higher mass biomolecules (e.g. proteins).

Na/K-ATPase as a target for anticancer drugs: studies with perillyl alcohol.

BACKGROUND: Na/K-ATPase (NKA) is inhibited by perillyl alcohol (POH), a monoterpene used in the treatment of tumors, including brain tumors. The NKA α1 subunit is known to be superexpressed in glioblastoma cells (GBM). This isoform is embedded in caveolar structures and is probably responsible for the signaling properties of NKA during apoptosis. In this work, we showed that POH acts in signaling cascades associated with NKA that control cell proliferation and/or cellular death. METHODS: NKA activity was measured by the amount of non-radioactive Rb(+) incorporation into cultured GBM cell lines (U87 and U251) and non-tumor cells (mouse astrocytes and VERO cells). Cell viability was measured by lactate dehydrogenase levels in the supernatants of POH-treated cells. Activated c-Jun N-terminal Kinase (JNK) and p38 were assessed by western blotting. Apoptosis was detected by flow cytometry and immunocytochemistry, and the release of interleukins was measured by ELISA. RESULTS: All four cell types tested showed a similar sensitivity for POH. Perillic acid (PA), the main metabolite of POH, did not show any effect on these cells. Though the cell viability decreased in a dose-dependent manner when cells were treated with POH, the maximum cytotoxic effect of PA obtained was 30% at 4 mM. 1.5 mM POH activated p38 in U87 cells and JNK in both U87 and U251 cells as well as mouse astrocytes. Dasatinib (an inhibitor of the Src kinase family) and methyl ß-cyclodextrin (which promotes cholesterol depletion in cell membranes) reduced the POH-induced activation of JNK1/2 in U87 cells, indicating that the NKA-Src complex participates in this mechanism. Inhibition of JNK1/2 by the JNK inhibitor V reduced the apoptosis of GBM cells that resulted from POH administration, indicating the involvement of JNK1/2 in programmed cell death. 1.5 mM POH increased the production of interleukin IL-8 in the U251 cell supernatant, which may indicate a possible strategy by which cells avoid the cytotoxic effects of POH. CONCLUSIONS: A signaling mechanism mediated by NKA may have an important role in the anti-tumor action of POH in GBM cells.

Na/K-ATPase assay in the intact mice lung subjected to perfusion.

BACKGROUND: Among the characteristics of acute respiratory distress syndrome (ARDS) is edema formation and its resolution depends on pneumocyte Na/K-ATPase activity. Increased concentration of oleic acid (OA) in plasma induces lung injury by targeting Na/K-ATPase and, thus, interfering in sodium transport. FINDINGS: Presently, we adapted a radioactivity-free assay to detect Na/K-ATPase activity in perfused lung mice, comparing the inhibitory effect of ouabain and OA. We managed to perfuse only the lung, avoiding the systemic loss of rubidium. Rb+ incorporation into lung was measured by inductively coupled plasma optical emission spectrometry (ICP OES) technique, after lung tissue digestion. Na/K-ATPase activity was the difference between Rb+ incorporation with or without ouabain. Lung Na/K-ATPase was completely inhibited by perfusion with ouabain. However, OA caused a partial inhibition. CONCLUSIONS: In the present work the amount of incorporated Rb+ was greater than seen in our previous report, showing that the present technique is trustworthy. This new proposed assay may allow researchers to study the importance of Na/K-ATPase activity in lung pathophysiology.

Murine lung injury caused by Leptospira interrogans glycolipoprotein, a specific Na/K-ATPase inhibitor.

BACKGROUND: Leptospiral glycolipoprotein (GLP) is a potent and specific Na/K-ATPase inhibitor. Severe pulmonary form of leptospirosis is characterized by edema, inflammation and intra-alveolar hemorrhage having a dismal prognosis. Resolution of edema and inflammation determines the outcome of lung injury. Na/K-ATPase activity is responsible for edema clearance. This enzyme works as a cell receptor that triggers activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathway. Therefore, injection of GLP into lungs induces injury by triggering inflammation. METHODS: We injected GLP and ouabain, into mice lungs and compared their effects. Bronchoalveolar lavage fluid (BALF) was collected for cell and lipid body counting and measurement of protein and lipid mediators (PGE2 and LTB4). The levels of the IL-6, TNFα, IL-1B and MIP-1α were also quantified. Lung images illustrate the injury and whole-body plethysmography was performed to assay lung function. We used Toll-like receptor 4 (TLR4) knockout mice to evaluate leptospiral GLP-induced lung injury. Na/K-ATPase activity was determined in lung cells by nonradioactive rubidium incorporation. We analyzed MAPK p38 activation in lung and in epithelial and endothelial cells. RESULTS: Leptospiral GLP and ouabain induced lung edema, cell migration and activation, production of lipid mediators and cytokines and hemorrhage. They induced lung function alterations and inhibited rubidium incorporation. Using TLR4 knockout mice, we showed that the GLP action was not dependent on TLR4 activation. GLP activated of p38 and enhanced cytokine production in cell cultures which was reversed by a selective p38 inhibitor. CONCLUSIONS: GLP and ouabain induced lung injury, as evidenced by increased lung inflammation and hemorrhage. To our knowledge, this is the first report showing GLP induces lung injury. GLP and ouabain are Na/K-ATPase targets, triggering intracellular signaling pathways. We showed p38 activation by GLP-induced lung injury, which was may be linked to Na/K-ATPase inhibition. Lung inflammation induced by GLP was not dependent on TLR4 activation.

Oleic acid inhibits lung Na/K-ATPase in mice and induces injury with lipid body formation in leukocytes and eicosanoid production.

BACKGROUND: Acute respiratory distress syndrome (ARDS) can emerge from certain pathologies, such as sepsis, fat embolism and leptospirosis, in which the levels of unesterified fatty acids are increased in the patient's plasma. ARDS is characterized by edema formation, and edema resolution occurs mainly due to the pneumocyte Na/K-ATPase activity. As previously described, increased oleic acid (OA) plasma concentrations induce lung injury by interfering with sodium transport. The first aim of this study was to develop a radioactivity-free assay to detect Na,K-ATPase activity ex vivo using a model of OA-induced lung injury in mice. We also investigated the relationship between Na/K-ATPase inhibition and OA-induced lung injury using ouabain-induced lung injury as a comparison, because of the well-described effect of ouabain as a Na/K-ATPase inhibitor. METHODS: We developed a Na/K-ATPase assay based on the capture of non-radioactive Rb+ ions by mice lung tissue in the absence or presence of ouabain, a specific Na/K-ATPase inhibitor. Rb+ incorporation into the lung was measured by inductively coupled plasma-optical emission spectrometry (ICP-OES) after lung tissue mineralization. Na/K-ATPase activity was considered as the difference between Rb+ incorporation in the absence and in the presence of ouabain. Bronchoalveolar lavage fluid was collected for lung injury assessment. For this assessment, cell counting, lipid body enumeration and lipid mediator concentrations were measured. Histological analyses were used to determinate lung pathology. Whole body plethysmographic analysis was performed to assay lung function. RESULTS: The lung Na/K-ATPase activity of mice was completely inhibited by an OA dose of 10 µmol, an effect also obtained with 10-3 µmol of ouabain, as demonstrated by the decreased Rb+ incorporation in the lungs. The same OA dose induced lung edema and inflammation with cell influx, lipid body formation, and leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) production. Ouabain also induced lung inflammation, as detected by histological examinations. As far as we know, this is the first time that ouabain-induced lung injury was shown. Both OA and ouabain induced functional lung pathology in mice simultaneously with inhibition of the lung Na/K-ATPase activity. CONCLUSIONS: We developed a new non-radioactive assay to quantified Na/K-ATPase in vivo. OA and ouabain inhibited in vivo Na/K-ATPase activity in the lungs and induced lung injury. Our data reinforce the idea that Na/K-ATPase inhibitors may worsen lung injury in specific pathological conditions.