981 resultados para Pompe sodium-potassium ATP dépendante


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1. In a fourway double-blind placebo controlled study, the effects of cilazapril, a new angiotensin converting enzyme inhibitor, on renal function and the responses to intravenous frusemide were studied in a group of twelve salt depleted male volunteers. 2. Cilazapril produced an increase in effective renal plasma flow and urinary output of prostaglandin E2 metabolite (PGE2-M) but no effect on sodium, potassium or water excretion. 3. Pretreatment with cilazapril antagonised the effects of frusemide on glomerular filtration, PGE2-M and sodium excretion.

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Tese de doutoramento, Medicina (Neurologia), Universidade de Lisboa, Faculdade de Medicina, 2015

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A flow injection analysis (FIA) system comprising a cysteine selective electrode as detection system was developed for determination of this amino acid in pharmaceuticals. Several electrodes were constructed for this purpose, having PVC membranes with different ionic exchangers and mediator solvents. Better working characteristics were attained with membranes comprising o-nitrophenyl octyl ether as mediator solvent and a tetraphenylborate based ionic-sensor. Injection of 500 µL standard solutions into an ionic strength adjuster carrier (3x10-3 M) of barium chloride flowing at 2.4mL min-1, showed linearity ranges from 5.0x10-5 to 5.0x10-3 M, with slopes of 76.4±0.6mV decade-1 and R2>0.9935. Slope decreased significantly under the requirement of a pH adjustment, selected at 4.5. Interference of several compounds (sodium, potassium, magnesium, barium, glucose, fructose, and sucrose) was estimated by potentiometric selectivity coefficients and considered negligible. Analysis of real samples were performed and considered accurate, with a relative error to an independent method of +2.7%.

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Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

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The membrane organization of the alpha-subunit of purified (Na+ + K+)-ATPase ((Na+ + K+)-dependent adenosine triphosphate phosphorylase, EC 3.6.1.3) and of the microsomal enzyme of the kidney of the toad Bufo marinus was compared by using controlled trypsinolysis. With both enzyme preparations, digestions performed in the presence of Na+ yielded a 73 kDa fragment and in the presence of K+ a 56 kDa, a 40 kDa and small amounts of a 83 kDa fragment from the 96 kDa alpha-subunit. In contrast to mammalian preparations (Jørgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415), trypsinolysis of the purified amphibian enzyme led to a biphasic loss of (Na+ + K+)-ATPase activity in the presence of both Na+ and K+. These data could be correlated with an early rapid cleavage of 3 kDa from the alpha-subunit in both ionic conditions and a slower degradation of the remaining 93 kDa polypeptide. On the other hand, in the microsomal enzyme, a 3 kDa shift of the alpha-subunit could only be produced in the presence of Na+. Our data indicate that (1) purification of the amphibian enzyme with detergent does not influence the overall topology of the alpha-subunit but produces a distinct structural alteration of its N-terminus and (2) the amphibian kidney enzyme responds to cations with similar conformational transitions as the mammalian kidney enzyme. In addition, anti alpha-serum used on digested enzyme samples revealed on immunoblots that the 40 kDa fragment was better recognized than the 56 kDa fragment. It is concluded that the NH2-terminal of the alpha-subunit contains more antigenic sites than the COOH-terminal domain in agreement with the results of Farley et al. (Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906).

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Cirrhosis is a frequent and severe disease, complicated by renal sodium retention leading to ascites and oedema. A better understanding of the complex mechanisms responsible for renal sodium handling could improve clinical management of sodium retention. Our aim was to determine the importance of the amiloride-sensitive epithelial sodium channel (ENaC) in collecting ducts in compensate and decompensate cirrhosis. Bile duct ligation was performed in control mice (CTL) and collecting duct-specific αENaC knockout (KO) mice, and ascites development, aldosterone plasma concentration, urinary sodium/potassium ratio and sodium transporter expression were compared. Disruption of ENaC in collecting ducts (CDs) did not alter ascites development, urinary sodium/potassium ratio, plasma aldosterone concentrations or Na,K-ATPase abundance in CCDs. Total αENaC abundance in whole kidney increased in cirrhotic mice of both genotypes and cleaved forms of α and γ ENaC increased only in ascitic mice of both genotypes. The sodium chloride cotransporter (NCC) abundance was lower in non-ascitic KO, compared to non-ascitic CTL, and increased when ascites appeared. In ascitic mice, the lack of αENaC in CDs induced an upregulation of total ENaC and NCC and correlated with the cleavage of ENaC subunits. This revealed compensatory mechanisms which could also take place when treating the patients with diuretics. These compensatory mechanisms should be considered for future development of therapeutic strategies.

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This investigation has three purposes I to make a comparative chemical study on sediment cores collected for Lake Lisgar (man-made lake in an urban center) and Lake Hunger (natural basin in a rural community) encompassing the time since European settlement I to determine the postglacial chemical history of Lake Hunger, and to determine the vegetational history of the Lake Hunger area from postglacial time to the present. The minus 80 mesh fraction of 108 soil samples and 18 stream sediment samples collected in the vicinity of Lakes' Lisgar and Hunger were analyzed for cold hydrochloric acid soluble lead, zinc, nickel, cobalt, copper, aluminum, sodium, potassium, calcium, magnesium, iron and manganese. Lacustrine sediments from 5 boreholes in the Lake Lisgar basin were collected. Boreholes 1, 2, 3, and 4 were analyzed for palynological and chemical information and Borehole 5 was subjected to pollen and ostracode analysis. Lacustrine sediments from 6 boreholes in the Lake Hunger basin were collected. Palyno- -logical and chemical analysis were performed on Boreholes 1, 2, 3, 4, and 6 and Borehole 5 was analyzed for pollen. In addition, radiocarbon dates were obtained on sediment samples from Boreholes 4 and 5. A total of 8 surface samples were collected from the margins of the Lake Hunger basin and these were chemically analyzed in the laboratory. All of the lacustrine sediments were ashed and analyzed for cold hydrochloric acid soluble lead, zinc, nickel, cobalt, copper, aluminum, sodium, potassium, calcium, magnesium, iron and manganese using a Perkin Elmer 40) Atomic Absorption spectrophotometer. The results . obtained for the 12 elements were expressed as parts per million in dry sediments. It was found that man's influence on the element distribution patterns in the sediments of Lake Lisgar appeared to be related to his urbanizing developments within the lake vicinity, whereas, the rural developments in the vicinity of lake Hunger appeared to have had little effect on the element distribution patterns in the lake sediments. The distribution patterns of lead, zinc, nickel, cobalt, aluminum, magnesium, sodium and potassium are similar to the % ash curve throughout postglacial time indicating that the rate of erosion in the drainage basin is the main factor which controls the concentration of these elements in the sediments of Lake Hunger. The vegetational history, from palynological analysis, of Lake Hunger from postglacial time to the present includes the following stages: tundra, open spruce forest, closed boreal forest, deciduous forest and the trend towards the re-establishment of pine following the clearing of land and the subsequent settlement of the Lake Hunger area by European settlers. The concentrations of some elements (cobalt, nickel, iron, manganese, calcium, magnesium, sodium and potassium) in the sediments of Lake Hunger appears to be higher during pre-cultural compared to post-cultural times. At least one complete postglacial record of the chemical history within a lake basin is necessary in order to accurately assess man's effects on his environment.

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Interactions of photoperiod and temperature upon waterelectrolyte balance were examined in rainbow trout acclimated to six combinations of two photoperiods {18h light: 6h dark, o 6h light: l8h dark) and three temperatures (2, 10 and 18 C). The influence of temperature and photoperiod upon plasma, skeletal muscle, cardiac muscle and liver levels of sodium, potassium, magnesi.um, calcium, chloride, water content, water distribution and cellular ion concentrations was determined by a one way analysis of variance. Significant (p < 0.05 or better) temperature effects at common photoperiods were observed in 70% of the analyses performed, showing no bias toward either photoperiod. Significant photoperiod effects occured in 57% of the analyses performed at common temperatures. The influence of photoperiod was most prevalent at reduced temperatures. Potassium and magnesium appeared to be particularly thermosensitive, while sodium and calcium were the most photosensitive of the electrolytes. The ionic composition of all tissues studied were relatively thermosensitive, with liver apparently being the most sensitive. On the other hand; the ionic composition of skeletal and cardiac muscle appear to be the mos.t photosensitive of the tissues examined. Water content and distribution in skeletal muscle and liver were significantly influenced by temperature in 50% of the analyses performed showing a very strong bias toward UwinterU animals. Photoperiod effects were significant in 56% of the water parameters measured with a strong bias toward the two lower temperatures. Body weight was of significant influence in 16% of the 174 analyses performed. These data are discussed in terms of the effect of temperature upon ionregulatory mechanisms and the possible impact of photoperiod variations on endocrine systems influencing water-electrolyte metabolism.

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Two groups of rainbow trout were acclimated to 20 , 100 , and 18 o C. Plasma sodium, potassium, and chloride levels were determined for both. One group was employed in the estimation of branchial and renal (Na+-K+)-stimulated, (HC0 3-)-stimulated, and CMg++)-dependent ATPase activities, while the other was used in the measurement of carbonic anhydrase activity in the blood, gill and kidney. Assays were conducted using two incubation temperature schemes. One provided for incubation of all preparations at a common temperature of 2S oC, a value equivalent to the upper incipient lethal level for this species. In the other procedure the preparations were incubated at the appropriate acclimation temperature of the sampled fish. Trout were able to maintain plasma sodium and chloride levels essentially constant over the temperature range employed. The different incubation temperature protocols produced different levels of activity, and, in some cases, contrary trends with respect to acclimation temperature. This information was discussed in relation to previous work on gill and kidney. The standing-gradient flow hypothesis was discussed with reference to the structure of the chloride cell, known thermallyinduced changes in ion uptake, and the enzyme activities obtained in this study. Modifications of the model of gill lon uptake suggested by Maetz (1971) were proposed; high and low temperature models resulting. In short, ion transport at the gill at low temperatures appears to involve sodium and chloride 2 uptake by heteroionic exchange mechanisms working in association w.lth ca.rbonlc anhydrase. G.l ll ( Na + -K + ) -ATPase and erythrocyte carbonic anhydrase seem to provide the supplemental uptake required at higher temperatures. It appears that the kidney is prominent in ion transport at low temperatures while the gill is more important at high temperatures. 3 Linear regression analyses involving weight, plasma ion levels, and enzyme activities indicated several trends, the most significant being the interrelationship observed between plasma sodium and chloride. This, and other data obtained in the study was considered in light of the theory that a link exists between plasma sodium and chloride regulatory mechanisms.

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Landscape geochemical investigations were conducted upon portions of a natural uniform landscape in southern Norway. This consisted of sampling both soil profile samples and spruce tree twigs for the analysis of twelve chemical elements. These elements were cobalt, copper, nickel, lead, zinc, manganese, magnesium, iron, calcium, sodium, potassium and aluminum which were determined by atomic absorption analysis on standardized extraction techniques for both organic and inorganic materials. Two "landscape traverses" were chosen for a comparative study of the effects of varying landscape parameters upon the trace element distribution patterns throughout the landscape traverses. The object of this study was to test this method of investigation and the concept of an ideal uniform landscape under Norwegian conditions. A "control traverse" was established to represent uniform landscape conditions typical of the study area and was used to determine "normal" or average trace element distribution patterns. A "signal traverse" was selected nearby over an area of lead mineralization where the depth to bedrock is very small. The signal traverse provided an area of similar landscape conditions to those of the control traverse with significant differences in the bedrock configuration and composition. This study was also to determine the effect of the bedrock mineralization upon the distribution patterns of the twelve chemical elements within the major components of the two landscape traverses (i.e. soil profiles and tree branches). The lead distribution within the soils of the signal traverse showed localized accumulations of lead within the overburden with maximum values occurring within the organic A horizon of soil profile #10. Above average concentrations of lead were common within the signal traverse, however, the other elements studied were not significantly different from those averages determined throughout the soils of the control traverse. The spruce twig samples did not have corresponding accumulations of lead near the soil lead anomaly. This is attributable to the very localized nature of the lead dispersion pattern within the soils. This approach to the study of the geochemistry of a natural landscape was effective in establishing: a) average or "normal" trace element distribution patterns b) local variations in the landscape morphology and c) the effect of unusually high lead concentrations upon the geochemistry of the landscape (i.e. within the soil profiles and tree branches). This type of study provides the basis for further more intensive studies and serves only as a first approximation of the behaviour of elements within a natural landscape.

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Le surenroulement de l’ADN est important pour tous les processus cellulaires qui requièrent la séparation des brins de l’ADN. Il est régulé par l’activité enzymatique des topoisomérases. La gyrase (gyrA et gyrB) utilise l’ATP pour introduire des supertours négatifs dans l’ADN, alors que la topoisomérase I (topA) et la topoisomérase IV (parC et parE) les éliminent. Les cellules déficientes pour la topoisomérase I sont viables si elles ont des mutations compensatoires dans un des gènes codant pour une sous-unité de la gyrase. Ces mutations réduisent le niveau de surenroulement négatif du chromosome et permettent la croissance bactérienne. Une de ces mutations engendre la production d'une gyrase thermosensible. L’activité de surenroulement de la gyrase en absence de la topoisomérase I cause l’accumulation d’ADN hyper-surenroulé négativement à cause de la formation de R-loops. La surproduction de la RNase HI (rnhA), une enzyme qui dégrade l’ARN des R-loops, permet de prévenir l’accumulation d’un excès de surenroulement négatif. En absence de RNase HI, des R-loops sont aussi formés et peuvent être utilisés pour déclencher la réplication de l’ADN indépendamment du système normal oriC/DnaA, un phénomène connu sous le nom de « constitutive stable DNA replication » (cSDR). Pour mieux comprendre le lien entre la formation de R-loops et l’excès de surenroulement négatif, nous avons construit un mutant conditionnel topA rnhA gyrB(Ts) avec l’expression inductible de la RNase HI à partir d’un plasmide. Nous avons trouvé que l’ADN des cellules de ce mutant était excessivement relâché au lieu d'être hypersurenroulé négativement en conditions de pénurie de RNase HI. La relaxation de l’ADN a été montrée comme étant indépendante de l'activité de la topoisomérase IV. Les cellules du triple mutant topA rnhA gyrB(Ts) forment de très longs filaments remplis d’ADN, montrant ainsi un défaut de ségrégation des chromosomes. La surproduction de la topoisomérase III (topB), une enzyme qui peut effectuer la décaténation de l’ADN, a corrigé les problèmes de ségrégation sans toutefois restaurer le niveau de surenroulement de l’ADN. Nous avons constaté que des extraits protéiques du mutant topA rnhA gyrB(Ts) pouvaient inhiber l’activité de surenroulement négatif de la gyrase dans des extraits d’une souche sauvage, suggérant ainsi que la pénurie de RNase HI avait déclenché une réponse cellulaire d’inhibition de cette activité de la gyrase. De plus, des expériences in vivo et in vitro ont montré qu’en absence de RNase HI, l’activité ATP-dépendante de surenroulement négatif de la gyrase était inhibée, alors que l’activité ATP-indépendante de cette enzyme demeurait intacte. Des suppresseurs extragéniques du défaut de croissance du triple mutant topA rnhA gyrB(Ts) qui corrigent également les problèmes de surenroulement et de ségrégation des chromosomes ont pour la plupart été cartographiés dans des gènes impliqués dans la réplication de l’ADN, le métabolisme des R-loops, ou la formation de fimbriae. La deuxième partie de ce projet avait pour but de comprendre les rôles des topoisomérases de type IA (topoisomérase I et topoisomérase III) dans la ségrégation et la stabilité du génome de Escherichia coli. Pour étudier ces rôles, nous avons utilisé des approches de génétique combinées avec la cytométrie en flux, l’analyse de type Western blot et la microscopie. Nous avons constaté que le phénotype Par- et les défauts de ségrégation des chromosomes d’un mutant gyrB(Ts) avaient été corrigés en inactivant topA, mais uniquement en présence du gène topB. En outre, nous avons démontré que la surproduction de la topoisomérase III pouvait corriger le phénotype Par- du mutant gyrB(Ts) sans toutefois corriger les défauts de croissance de ce dernier. La surproduction de topoisomérase IV, enzyme responsable de la décaténation des chromosomes chez E. coli, ne pouvait pas remplacer la topoisomérase III. Nos résultats suggèrent que les topoisomérases de type IA jouent un rôle important dans la ségrégation des chromosomes lorsque la gyrase est inefficace. Pour étudier le rôle des topoisomérases de type IA dans la stabilité du génome, la troisième partie du projet, nous avons utilisé des approches génétiques combinées avec des tests de « spot » et la microscopie. Nous avons constaté que les cellules déficientes en topoisomérase I avaient des défauts de ségrégation de chromosomes et de croissance liés à un excès de surenroulement négatif, et que ces défauts pouvaient être corrigés en inactivant recQ, recA ou par la surproduction de la topoisomérase III. Le suppresseur extragénique oriC15::aph isolé dans la première partie du projet pouvait également corriger ces problèmes. Les cellules déficientes en topoisomérases de type IA formaient des très longs filaments remplis d’ADN d’apparence diffuse et réparti inégalement dans la cellule. Ces phénotypes pouvaient être partiellement corrigés par la surproduction de la RNase HI ou en inactivant recA, ou encore par des suppresseurs isolés dans la première partie du projet et impliques dans le cSDR (dnaT18::aph et rne59::aph). Donc, dans E. coli, les topoisomérases de type IA jouent un rôle dans la stabilité du génome en inhibant la réplication inappropriée à partir de oriC et de R-loops, et en empêchant les défauts de ségrégation liés à la recombinaison RecA-dépendante, par leur action avec RecQ. Les travaux rapportés ici révèlent que la réplication inappropriée et dérégulée est une source majeure de l’instabilité génomique. Empêcher la réplication inappropriée permet la ségrégation des chromosomes et le maintien d’un génome stable. La RNase HI et les topoisomérases de type IA jouent un rôle majeur dans la prévention de la réplication inappropriée. La RNase HI réalise cette tâche en modulant l’activité de surenroulement ATP-dependante de la gyrase, et en empêchant la réplication à partir des R-loops. Les topoisomérases de type IA assurent le maintien de la stabilité du génome en empêchant la réplication inappropriée à partir de oriC et des R-loops et en agissant avec RecQ pour résoudre des intermédiaires de recombinaison RecA-dépendants afin de permettre la ségrégation des chromosomes.

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The present study addresses to understand the sedimentological properties of the coasts of kodungallur and chellanam, central Kerala to bring out the relationship between the textural, mineralogical and geochemical characters with that of the respective environment. The grain size study of the beach ridge sediments from different pits has been investigated at close intervals, which enables to understand the grain size variations with depth. The sediment samples from various pits of the beach ridges indicate that the sediments range primarily from medium to very fine sand, well to moderately sorted, fine to coarse skewed and leptokurtic to platykurtic. The study area is considered as a prograding coast. Variations in grain size down the pit give three phases of beach building activities i.e.; a coarsening upward sequence in the bottom layers, a fining upward in the middle and coarsening upward in the top. Beach ridges are formed by swash built sediments with cross bedding and setting lag type sediments with seaward dipping/horizontal units. Geochemical signatures in the study area have been brought out through the analysis of major and trace elements. Iron is significantly enriched and its control over many trace elements is evident. Copper, chromium, cobalt, lithium, lead and zinc show decreasing trend with depth, while sodium, potassium,strontium,nickel and organic carbon increases. The association of many trace elements with organic carbon has also been established. Dissolution of trace elements in anoxic environment, at depth and reprecipitation in the oxic layers, at near or subsurface, are the major mechanism that brought out the variation of certain environmentally sensitive elements

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In der vorliegenden Arbeit wurde gezeigt, wie mit Hilfe der atomaren Vielteilchenstörungstheorie totale Energien und auch Anregungsenergien von Atomen und Ionen berechnet werden können. Dabei war es zunächst erforderlich, die Störungsreihen mit Hilfe computeralgebraischer Methoden herzuleiten. Mit Hilfe des hierbei entwickelten Maple-Programmpaketes APEX wurde dies für geschlossenschalige Systeme und Systeme mit einem aktiven Elektron bzw. Loch bis zur vierten Ordnung durchgeführt, wobei die entsprechenden Terme aufgrund ihrer großen Anzahl hier nicht wiedergegeben werden konnten. Als nächster Schritt erfolgte die analytische Winkelreduktion unter Anwendung des Maple-Programmpaketes RACAH, was zu diesem Zwecke entsprechend angepasst und weiterentwickelt wurde. Erst hier wurde von der Kugelsymmetrie des atomaren Referenzzustandes Gebrauch gemacht. Eine erhebliche Vereinfachung der Störungsterme war die Folge. Der zweite Teil dieser Arbeit befasst sich mit der numerischen Auswertung der bisher rein analytisch behandelten Störungsreihen. Dazu wurde, aufbauend auf dem Fortran-Programmpaket Ratip, ein Dirac-Fock-Programm für geschlossenschalige Systeme entwickelt, welches auf der in Kapitel 3 dargestellen Matrix-Dirac-Fock-Methode beruht. Innerhalb dieser Umgebung war es nun möglich, die Störungsterme numerisch auszuwerten. Dabei zeigte sich schnell, dass dies nur dann in einem angemessenen Zeitrahmen stattfinden kann, wenn die entsprechenden Radialintegrale im Hauptspeicher des Computers gehalten werden. Wegen der sehr hohen Anzahl dieser Integrale stellte dies auch hohe Ansprüche an die verwendete Hardware. Das war auch insbesondere der Grund dafür, dass die Korrekturen dritter Ordnung nur teilweise und die vierter Ordnung gar nicht berechnet werden konnten. Schließlich wurden die Korrelationsenergien He-artiger Systeme sowie von Neon, Argon und Quecksilber berechnet und mit Literaturwerten verglichen. Außerdem wurden noch Li-artige Systeme, Natrium, Kalium und Thallium untersucht, wobei hier die niedrigsten Zustände des Valenzelektrons betrachtet wurden. Die Ionisierungsenergien der superschweren Elemente 113 und 119 bilden den Abschluss dieser Arbeit.

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A bioimpedância parece ser especialmente adequada à medição não-invasiva de diversas variáveis fisiológicas. O presente estudo pretendeu averiguar se a quantificação da água corporal total e dos compartimentos intra e extracelular, obtida com um equipamento de análise baseada na bioimpedância, é relacionável com os valores das concentrações dos iões sódio, potássio e cloreto obtido a partir do ionograma do sangue venoso. Foram estudados 12 voluntários, de ambos os géneros, idade e condição de saúde variadas, embora sem qualquer patologia do foro hidro-electrolítico. As medições da água corporal foram obtidas com o Bodystat® QuadScan 4000 e o ionograma por potenciometria. Nas presentes condições experimentais, que apenas devem ser entendidas como uma abordagem preliminar às questões de investigação levantadas, não foram demonstradas relações entre as variáveis em estudo. Contudo, o interesse potencial e a praticabilidade do método, justificam novas abordagem que visem aumentar o número de paciente estudados e refinar a metodologia de análise.