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Estimation of extracellular space and blood volume using bioelectrical impedance measurements.

The bromide-82 dilution space (extracellular space, ECS) and blood volume (BV) were measured in 21 patients with esophageal and gastric cancer and in 27 patients 18-96 months after total gastrectomy. Resistance (R) and reactance (Xc) from bioelectrical impedance measurements were used to obtain multiple regression equations for ECS and BV. The variables weight, gender, and height 2/Xc were independent predictors of ECS (r = 0.767; P less than 0.0001). Height 2/R and gender were predictors of blood volume (r = 0.856; P less than 0.0001). The mean difference between the Br space and the ECS predicted from impedance measurements was 0 +/- 1.54 (mean +/- SD). The limits of agreement (+/- 2 SD) were therefore +/- 3.08 l or 19.6% of the mean Br space of 15.7 l. The limits of agreement for BV were +/- 789 ml or +/- 19.7% of the average BV of 4008 ml. It is concluded that bioelectrical impedance plethysmography using a single frequency can be used for the estimation of ECS and BV. The wide limits of agreement, however, may limit its used in clinical practice.

Adult

Fractional extracellular space and fractional water content of various rat tissues at different extracellular pH values and in uremia.

At different extracellular pH values fractional extracellular space (calculated from the distribution of 3H inulin) and fractional water content showed no significant differences. 72 h after nephrectomy both variables increased significantly. An exception to this was brain, where fractional extracellular space decreased and total intracellular water increased as a sign of brain oedema.

Animals

Influence of contractile state on the size of the extracellular space in isolated ventricular myocardium.

A method is described with which the extracellular space of isolated ventricular muscle can be measured accurately and continuously in the same muscle. The size of the extracellular space is shown to vary with the contractile state of the myocardium. Interventions such as quiescence, manganese and acidosis reduce myocardial contractility and increase the size of the extracellular space. Barium, ouabain and hypoxia cause contracture and reduce the size of the extracellular space. These changes should be taken into account when measuring intracellular electrolytes in isolated ventricular preparations.

Acidosis, Respiratory

The behaviour of different markers of the mucosal extracellular space in rat small intestine.

The mucosal extracellular space in rat small intestine is determined with inulin-14C, mannitol-14C and unlabeled 2-deoxy-D-glucose in control animals and after an intravenous secretin load. The mannitol and 2-deoxy-D-glucose spaces in control animals are significantly greater than the inulin space. After secretin the three investigated markers show different, in part adverse, alterations of the mucosal extracellular space. These different results may be due to the composition of the incubation medium and to a distinct effect of secretin on the distribution of the employed markers.

Animals

Release of enzymes from cells: transport and distribution within the extracellular space.

The distribution in the extracellular space of enzymes released from organ cells was investigated using three models: (1) comparison of enzyme activities in blood plasma and lymph of the ductus thoracicus (dog) and plasma and intestinal lymph (rat); (2) i.v. injection of heterologous, homologous and autologous enzymes in order to increase acutely the activities and to measure the rate constants for the distribution and elimination of the enzymes (rat); or (3) plasmapheresis in order to create an enzyme activity gradient from the interstitial space and to determine the rate constants for the reestablishment of the equilibrium between the extra and intravascular compartments (rat). The results suggest that the enzymes are mainly released into the interstitial fluid and transported via the lymph into the intravascular compartment. From there the enzymes diffuse back into the interstitial compartment and are eliminated by a yet unknown mechanism. Transport of enzymes across the capillary membranes in both directions depends on (1) the permeability of the capillary membranes, which varies from region to region and (2) the molecular seizes of the enzymes.

Animals

Brain extracellular space fixed for electron microscopy.

Adult mammalian brain contains 17--20% extracellular space, but fixatives cause the cellular elements to ingest the extracellular fluid so that the space is reduced to less than 5% with all conventional methods of fixation. This can be prevented by washing out the extracellular fluid with isotonic sucrose, which does not penetrate cells. Subsequent fixation with aldehydes and osmium and conventional processing leads to the preservation of extracellular space in electron micrographs. Extracellular space was found to be unevenly distributed, widely separating some cellular processes while leaving other groups of processes contiguous.

Animals

Age-related change in the neuronal microenvironment: penetration of ruthenium red into extracellular space of brain in young adult and senescent rats.

The volume of the extracellular space, which contributes to the microenvironment of neurons, is diminished in the brains of senescent (as compared to adult) rats and an age-related change in its composition has been hypothesized. To test this hypothesis we have compared the penetration of ruthenium red, a polyanion selectively distributed in the extracellular space, into the dentate gyri of young adult and senescent Fischer 344 rats. Slices of hoppocampal formation were fixed by immersion, first in a glutaraldehyde solution containing ruthenium red, then in a solution of osmium tetroxide containind examined by electron microscopy. Dense particles of ruthenium red reaction product were readily localized in intercellular channels and synaptic clefts and the depth of penetration of ruthenium red in 25-month-old rats, as compared with 3-month-old animals, was found. These data indicate an age-related change in the charge density of the intercellular channels in the dentate gyrus of 25-month-old rats. They suggest a primary age-related change in the charg density of extracellular macromoledules, presumed to be primarily glycosaminoglycans, with a consequent change in water binding capacity and volume of the extracellular space.

Aging

[A synthetic review on the cerebral extracellular space evaluated by biochemical procedures (author's transl)].

The Authors present a synthetic review on the problem of the cerebral extracellular space evaluated by biochemical procedures. The studies in vitro are numerous, focused on various aspects, such as the choice of the marker, the influence of temperature, and that of the chemical composition of incubation media, the regional heterogeneity of the brain, and so on; it has been observed that inulin, which is the most commonly employed marker, gives an extracellular space of about 40-66%, whereas other markers give values higher than 45%; the variability of values depends on various experimental factors. The studies in vivo present some methodological difficulties due to the existence of the brain barrier; the most reliable procedure appears that of the continuous perfusion of the marker into the cerebrospinal fluid associated with a simultaneous intravenous perfusion; this procedure gives quantitative estimates of extracellular space of about 14-30%. Taking into account that other experimental methods (such as the morphological and the electrophysiological) give lower values, respectively of 5-20% and 18-25%, the Authors found that it should be possible to agree about an average value of cerebral extracellular space of 15-20%. This appears to be of some relevance especially in studying the pathological problems of the brain, and particularly cerebral edema.

Brain

Potassium activity in photoreceptors, glial cells and extracellular space in the drone retina: changes during photostimulation.

1. A double-barrelled potassium-sensitive micro-electrode was developed that was fine enough to record intracellular electrical potentials and potassium activities (aK) in the drone retina. 2. aK was measured in the photoreceptor cells, in the pigment (glial) cells, and in the extracellular space, in the superfused, cut, retina. The effect of photostimulation was studied: 20 msec light flashes, intense enough to evoke receptor potentials of maximum amplitude were presented, 1/sec, in a train lasting about 2 min. 3. In photoreceptors with membrane potentials greater than or equal to 50 mV aK in the dark was 79 mM, S.D. = 27 mM, n = 11. During photostimulation aK fell by 21.5 +/- 9.5 mM with a half-time of 30 +/- 22 sec. (A tentative conversion from activities to free concentrations can be made by taking the activity coefficient as 0.70 its value in the Ringer solution). 4. In pigment cells with membrane potentials greater than or equal to 50 mV, aK in the dark was 52 mM, S.D. = 13 mM, n = 11. During photostimulation aK increased by 14 +/- 5 mM. 5. In the extracellular space aK increased during photostimulation with a mean half-time of less than 1.3 sec to a maximum (mean value 14 mM, S.D. = 8.4 mM, n = 22), and then fell to a plateau. 6. It is estimated from the anatomy that the photoreceptors occupy approximately 38% of the total volume of the retina, the pigment cells 57%, and extracellular space 5%. Hence, it seems possible that during photostimulation nearly all the net loss of potassium from the photoreceptors is temporarily stored in the pigment cells. 7. Recordings were made in the extracellular space of the intact animal by passing the electrode through a hole in the cornea. The mean aK in the dark was 7.7 mM, S.E. = 0.4 mM, n = 22. In the superfused retina, aK in the dark was 6.3 mM, S.E. = 0.7 mM, n = 22, even though aK in the Ringer solution was 2.2 mM. Increasing the aK of the Ringer solution to 7.0 mM had no apparent effect on aK in the extracellular space at depths greater than 20 micron. 8. In the intact animal the amplitude and time course of the change in extracellular aK evoked by the standard pattern of photostimulation were within the range observed in the superfused preparation.

Animals

Quantitative analysis of extracellular space using the method of TMA+ iontophoresis and the issue of TMA+ uptake.

The tetramethylammonium (TMA+) method for measuring the volume fraction and tortuosity of brain extracellular space is presented in detail. The temporal and spatial distribution of TMA+ in the extracellular space following iontophoresis or pressure microinjection is described by suitable equations and illustrated with graphs. By fitting the equations to the concentration versus time data obtained from measurements with ion-selective micropipettes, the volume fraction and tortuosity can be measured. In addition, the concentration-dependent uptake of TMA+ can be estimated from the given equations. The final section of the paper derives simple numerical estimates of TMA+ loss from the extracellular space by this mechanism.

Animals

Discrepancies in the extracellular space of sympathetic ganglia measured using different isotopes of mannitol and sucrose.

The extracellular space of rat superior cervical ganglia in vitro was measured using mannitol and sucrose labelled with tritium and carbon-14. The volumes of distribution of the 3H-labelled derivatives, especially [3H]mannitol, exceeded those of the 14C-derivatives. The divergence increased with increasing lengths of incubation. Thus, after 30 min incubation, 'spaces' (ml . g-1) were: [14Cu]mannitol, 0.407; [3H]mannitol 0.447; [14C]mannitol, 0.458; [3H]mannitol, 0.645; [14C]sucrose, 0.430; [3H]sucrose, 0.497. Using thin layer chromatography, it was shown that an average of 22% of the label in ganglia incubated for 120 min with [3H]mannitol, but only 4% with [14C]mannitol, was not associated with the parent compound. Both [3H]- and [14C]sucrose appeared to be metabolized by 11%. It is concluded that mannitol and sucrose can be metabolized in isolated ganglia and that this may lead to substantial errors in estimating the extracellular space, particularly when [3H]markers are used.

Animals

Clearance of brain edema and macromolecules through the cortical extracellular space.

The transit routes of fluid and particulate matter through brain tissue remain unclear. The object of this study was to examine the movement of macromolecules through brain tissue to further clarify the clearance pathways of edema proteins as they migrate toward the cortex. For this purpose, albumin solution (20 microliters rat albumin diluted to 65 mg/ml with mock cerebrospinal fluid (CSF)) was intracerebrally infused into the caudate putamen, and the migration through brain tissue as well as through the ultrastructure of the cortical surfaces was explored using an immunocytochemical technique. The authors observed immunoreactive product on the glial limitans and pial lining as well as in the extracellular space of the cortical neuropil at 24 hours postinfusion, confirming that the protein had reached the cortical surface. To confirm the efflux of macromolecules into the subarachnoid CSF, 71,200 D fluorescein isothiocyanate-dextran (FITC-dextran 71,200) was infused; cortical surfaces of brains removed en bloc as well as coronal sections were macroscopically observed under ultraviolet illumination at 15 minutes and 24 hours postinfusion. It was observed that infused FITC-dextran 71,200 mainly localized in the cortical white matter and caudate putamen of the infusion site at 15 minutes postinfusion and by 24 hours was distributed in the entire cortex of the infused hemisphere. However, the dynamics of lower-molecular-weight substances was completely different. The spatial distribution of FITC-dextran 4400 diverged upward toward the cortical surface and spread more extensively than FITC-dextran 71,200. These observations were consistent with a diffusion process as the spread of the tracer was dependent upon molecular size. These studies provide compelling evidence that a process other than bulk flow was involved in the spread of macromolecules through the extracellular space of the normal cortical neuropil to sink into the subarachnoid space. It was concluded that the CSF pathway via the extracellular space of the cortical neuropil is a primary route for clearance of extracellular edema proteins to the subarachnoid space and that diffusion is involved in this process.

Albumins

Membrane potential and ion concentration stability conditions for a cell with a restricted extracellular space.

For an isolated membrane, the resting (zero current) potential is stable is the slope conductance is positive, and is unstable if the slope conductance is negative. Recent work suggests that the properties of many preparations are influenced by the presence of an extracellular space that is not in good diffusive contact with the bulk extracellular fluid. Ionic current flow across the membrane changes the ion concentrations in this space. These concentration changes affect the stability of the membrane potential. Even if the slope conductance is negative, the presence of the extracellular space can confer stability on the resting potential. Conversely, even if the slope conductance is positive, the extracellular space can produce instability of the resting potential. Evaluation of the relevant parameters for cardiac Purkinje fibres, from published experimental data, suggests that concentration changes in the extracellular space may play a significant role in determining when an action potential is initiated.

Animals

Extracellular space, water, and ion concentration in the hypertrophied rat myocardium.

Extracellular space (ECS), water, and intracellular ion concentration were determined in the normal and hypertrophied rat myocardium. Theoretical measurements of myocardial ECS based on a mathematical model (9.31%) closely approximated the observed results in the normal myocardium (12.8%) and hypertrophied myocardium (11.7%). Observed results confirmed theoretical considerations of no change in ECS with hypertrophy. ECS and water content were higher in the right ventricle than the left ventricle, but intracellular electrolytes remained unchanged. Myocardial hypertrophy did not alter these relationships.

Animals

The contribution of local blood flow to the rapid clearance of potassium from the cortical extracellular space.

The transport of potassium to the blood stream following stimulation of the cortex in cats is evaluated by means of a potassium sensitive microelectrode technique. Potassium levels are measured in cortical veins, the sagittal sinus and the extracellular space during and after both pharmacological and electrical stimulation of the cortex. It is concluded that the potassium transport to the blood stream is not a significant factor in the rapid clearance of potassium from the extracellular space following stimulation.

Animals

Extracellular space of frog skeletal muscle in vivo and in vitro: relation to proton magnetic resonance relaxation times.

1. The Na and Cl distribution spaces of freshly isolated frog muscles are 16.7 and 12.6%, respectively. These values increase to 25.6 and 23.3%, respectively, on incubation. 2. The extracellular components of both Na and Cl efflux curves are significantly smaller in freshly isolated muscles (approximately 12%) than in incubated muscles (approximately 18%). The fast exchanging A component of the extracellular space is increased more by incubation than the more slowly exchanging B component. 3. The proton magnetic resonance (p.m.r.) transverse relaxation curve for the water of freshly isolated frog muscles did not show the long, slowly relaxing tail present in curves from muscles incubated in Ringer solution. 4. When muscles were incubated in hypertonic solutions the p.m.r. transverse relaxation curves could be resolved into three components whose sizes were consistent with the components present in the sodium and chloride efflux curves. The non-exponentiality of the p.m.r. transverse relaxation curve therfore appears to arise from water in both the A and B extracellular compartments of muscle. 5. Efflux analysis indicated that the cellular Na content of both freshly isolated and incubated frog muscle is similar to that predicted by others (Lev, 1964; Armstrong & Lee, 1971; Lee & Armstrong, 1974) from measurements of intracellular Na ion activity using Na-sensitive micro-electrodes. The remainder of the tissue Na was found in the more rapidly exchanging extracellular compartments. The results of these experiments are inconsistent with the presence of a substantial fraction of bound Na in frog muscle. 6. These experiments show that muscle extracellular space is smaller in vivo than in vitro. Efflux analysis is suggested as the most accurate method of assessing extra-cellular components.

Animals

The determination of the myocardial extracellular space in the cat in vivo: a comparative methodological study.

The myocardial extracellular space (ECS) was determined in cats in vivo by means of the ECS indicators, inulin and sulfanilic acid and, additionally, as glucose space. Experiments were carried out in cats subjected to bilateral kidney ligation (single injection) and in intact cats (single injection or infusion of indicator). The heart was clamped by instant deep-freezing in situ; this technique was compared, in renally-ligated cats, with excision and subsequent freezing of the heart in liquid nitrogen. The myocardial blood content was significantly decreased, and the myocardial lactate concentration significantly increased in excised, as compared with in situ-clamped hearts. Renally-ligated cats showed marked hypotension. The mean myocardial blood content was also significantly lower than in intact animals. A highly significant correlation was found between myocardial blood content and blood pressure for all experiments with instant deep-freezing. The mean inulin ECS value in renally-ligated cats was 22.7 +/- 1.5 ml. In accordance with the fact that the ECS is dependent on the tissure blood content, the corresponding values in intact animals were significantly higher, 25.9 +/- 2.5 ml (single injection) and 26.3 +/- 3.9 ml (infusion), calculated per 100 g tissue wet weight. Similar values were obtained for the glucose and sulfanilic acid ECS. If the interstitial space, an expression independent of the tissue blood content is used as space parameter, no significant differences were found under any of the present experimental conditions.

Animals

[The development of intramyocardial haemorrhages or fluidaccumulation in the extracellular space].

Isolated rat hearts according to Langendorff and rabbit hearts after orthostatic collapse were studied under the light and electron microscope. The light-micrographs were also quantitatively analysed. Changes in the vessels are noticeable, especially those in the sinusoid's. Vessel ruptures occur, also isolated endothelial ruptures with intact basement membranes and perfusion fluid enters the extracellular space. Numerous 0.06-01 mu large vesicles appear in the cytoplasma of the endothelial cells. A large number of vesiculation processes can be demonstrated along the cell membrane. The extracellular space is strongly-dilated. Changes in the colloid osmotic pressure and a rise in perfusion pressure have no clearly demonstrable influence on the extent of extravasation under the experimental conditions. The mechanism possible inducing such vessel changes is discussed.

Animals