Extracellular Space Marker Research Articles

Insulin regulates the paracellular permeability of cultured intestinal epithelial cell monolayers.

The T84 human colonic epithelial cell line retains the ability to produce secretagogue-responsive monolayer cultures with high transepithelial resistance when grown and maintained on collagen-coated permeable supports in media supplemented with 5% newborn calf serum. The addition of highly purified insulin to the basolateral but not the apical membrane side of established monolayers caused the transepithelial resistance to decline more than eightfold over a 3-4-d period. By comparing the transepithelial flux of 22Na with that of the extracellular space marker, [3H]mannitol, the decline in electrical resistance was shown to be due solely to an effect on tight junction-mediated paracellular permeability. The effect of insulin was dose dependent with a half-maximal effect at 3.9 ng/ml (approximately 0.7 nM) and fully reversible over a 10-d time course. Simultaneous addition of 2 microM cycloheximide prevented the insulin-induced decline in resistance; in fact, this combination caused a significant increase in electrical resistance. There was no effect on the short-circuit current response of insulin-treated monolayers to secretagogues so long as media was changed daily. While no gross morphological changes were apparent, there did appear to be a subtle condensation of the perijunctional actin ring as visualized using rhodamine-labeled phalloidin. These results demonstrate that insulin modulates the permeability of the occluding junction in T84 cell monolayers through a receptor mediated process which probably involves changes in protein synthesis and cytoskeletal structure. Insulin was also shown to produce similar effects on two other intestinal epithelial cell lines.

A correlation of permeabilities for passively transported compounds in monkey and rabbit jejunum.

Permeability measurements were conducted for a series of compounds using in vitro tissue sections from monkey and rabbit jejunum. Jejunal segments were stripped of serosal musculature and mounted in a diffusion-cell system, using previously described methods and equipment. Permeability determinations of radiolabeled compounds ranging over two orders of magnitude in molecular weight were conducted. For the compounds examined, the permeability of the rabbit jejunum was approximately twice that of the monkey. This was in contrast to the relationship implied by the stripped tissue thickness measurements of 0.92 and 0.83 mm for rabbit and monkey, respectively. An investigation of the size of the paracellular space in the jejunum was undertaken to account for this apparent discrepancy in tissue permeability. Scanning electron micrographs of intestinal sections revealed a similar packing density of cells between species; however, a difference was noted in the shape and number of villi per unit area. Comparative measurements of the paracellular volume in both species using mannitol and methoxyinulin as extracellular space markers further suggests that the paracellular junctions are similar in size but more numerous per unit area of rabbit jejunum than that of the monkey. In contrast to passively transported compounds, the active transport of D-glucose was greater in monkey jejunum compared to rabbit tissue segments. When active transport was inhibited by blockade of the sodium pump with ouabain, the passive component of D-glucose transport for both rabbit and monkey tissue was in agreement with the relationship demonstrated above for compounds which are solely transported by passive processes.

Freezing avoidance and the distribution of antifreeze glycopeptides in body fluids and tissues of Antarctic fish.

The distribution of antifreeze glycopeptides (AFGPs) in the body fluids and tissues of antarctic notothenioid fish was determined. In Dissostichus mawsoni (Norman), the peritoneal, pericardial and extradural fluid, like the blood, contained all eight AFGPs and in concentrations sufficient to depress freezing points below that of sea water (-1.9 degree C). Secreted fluids including urine, endolymph and aqueous and vitreous humour either lack all AFGPs or have very low concentrations of only the low molecular weight forms and have freezing points of about -1.0 degree C, and are therefore undercooled with respect to environmental temperature. Fluids with high concentrations of AFGPs also contain high levels of proteins similar to plasma proteins. Systemic administration of tritiated AFGPs in the closely related species Trematomus bernacchii (Boulenger) yielded a distribution pattern similar to that of the native AFGPs in D. mawsoni. This suggests passive distribution of AFGPs into the various fluid compartments following secretion from the liver; a pattern typical of secreted blood proteins. Tissue distribution of AFGPs was determined by comparison with that of the extracellular space marker [14C]polyethylene glycol. AFGPs were found in the interstitial fluid of all body tissues examined except brain tissue. No tissue showed any intracellular accumulation of tritiated AFGPs from the blood.

Improvement in myocardial performance without a decrease in high-energy phosphate metabolites after isoproterenol in Syrian cardiomyopathic hamsters.

To determine the effect of isoproterenol on cardiac energetics and function in an animal preparation of cardiomyopathy, we studied Langendorff perfused hearts from Syrian cardiomyopathic hamsters. High-energy phosphate metabolites (phosphocreatine [PCr], ATP, inorganic phosphate [Pi]) and intracellular pH (pHi) were measured by 31P nuclear magnetic resonance spectroscopy and correlated with left ventricular developed pressure, coronary flow, and O2 consumption before and during a 10(-6)M infusion of isoproterenol. Total intracellular calcium was also determined by atomic absorption spectroscopy with the use of potassium ethylenediamine tetra-acetate cobaltate as a marker for extracellular space. In cardiomyopathic hamsters, isoproterenol infusion increased mean developed pressure by 300% (p less than .005 compared with control; n = 5), O2 consumption eightfold (p less than .0005), and PCr by 40% (p less than .05). PCr/Pi ratio, which is analogous to phosphorylation potential, improved 100% (p = .05). In normal hamsters, isoproterenol infusion resulted in an 83% increase in developed pressure (p less than .001) and a 25% increase in O2 consumption (NS). However, mean PCr and PCr/Pi decreased by 30% and 50%, respectively (p less than .05 for both), during isoproterenol infusion. pHi decreased in normal animals (p less than .01), but tended to improve in diseased animals (NS) during isoproterenol infusion. Freeze-clamp measurements of phosphate metabolites correlated well with the nuclear magnetic resonance data. Intracellular calcium increased from 0.0102 +/- 0.002 to 0.144 +/- 0.030 mumol/ml heart water in normal hamsters during isoproterenol infusion. Cardiomyopathic hamsters had a markedly elevated baseline calcium content of 60.82 +/- 5.85 mumol/ml heart water due to the presence of dystrophic calcification.(ABSTRACT TRUNCATED AT 250 WORDS)

Origins of endogenous noradrenaline overflow during reperfusion of the ischaemic rat heart.

1. Reperfusion of the globally ischaemic isolated rat heart is associated with an enhanced overflow of endogenous noradrenaline (NA) after ischaemic periods of 20, 40 or 60 min but not of 10 min. 2. Reperfusion NA overflow, after 40 min of ischaemia, is suppressed by desipramine and increased when ischaemia follows a period of substrate deprivation. 3. Reperfusion after 40 min of ischaemia is associated with a significant rise in NA concentration despite a simultaneous 20-fold increase in flow. This increase in concentration is abolished by treatment with desipramine or if ischaemia follows a period of substrate deprivation. 4. Reperfusion NA overflow correlates with the reperfusion overflow of an extracellular space marker infused before the ischaemic episode. 5. These results suggest that ischaemia is heterogeneous and that NA is released into regions of particularly profound ischaemia from which it is subsequently eluted during reperfusion.

Trans-sarcolemmal Ca2+ movements associated with contraction of the rabbit right ventricular wall.

The purpose of this study was to examine the movements of Ca2+ into the myocardium from the extracellular space during the course of muscle contraction. Equilibration of the rabbit right ventricular wall with perfusate containing 45Ca was measured by collecting effluent droplets over time. This protocol was carried out in a quiescent muscle and then repeated 15-20 minutes later in an identical fashion except that halfway through the collection period the muscle was stimulated to contract. We were thus able to quantitate the contraction-dependent changes in 45Ca movements. In control experiments using [58Co]EDTA as an extracellular space marker, we observed that contraction altered the volume of this space. This alteration in extracellular space was relatively small, and the quantitation of 45Ca movements was corrected for this change. The addition of 1 microM Bay K 8644 to the perfusate stimulated tension and augmented Ca2+ depletion from the extracellular space. The addition of 15 microM nifedipine to the perfusate significantly reduced both tension development and Ca2+ depletion from the extracellular space of the muscle. Net contraction-dependent movement of Ca2+ from the extracellular space into the myocardium under control conditions at 1 mM [Ca2+] was 10-14 mumol Ca2+/kg tissue wet wt/beat. This value indicates either a large contribution of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum and/or significant contribution of sarcolemmal bound Ca2+ to excitation-contraction coupling in the rabbit ventricle.

Ascorbic acid: a nonradioactive extracellular space marker in canine heart.

The distribution pattern of ascorbic acid and L-[14C]ascorbic acid in myocardial tissue was compared with those of the classical radioactive extracellular space markers [3H]-inulin, [3H]sucrose, and Na82Br. A new polarographic technique was developed for analogue registration of ascorbic acid concentration in coronary venous blood. The kinetic data of the markers were studied in an open-chest canine heart preparation (n = 17 dogs) during a constant tracer infusion of up to 9 min. Distribution volumes were calculated based on the mean transit time method of Zierler (K. L. Zierler, Circ. Res. 10:393-407, 1962). The distribution volume of ascorbic acid (23.6 ml/100 g wet wt) as well as of L-[14C]ascorbic acid (24.5 ml/100 g wet wt) in myocardial tissue agreed closely with those of [3H]inulin (18.6 ml/100 g wet wt) and [3H]sucrose (22.0 ml/100 g wet wt), as well as 82Br- (27.3 ml/100 g wet wt). The obtained kinetic data confirmed that ascorbic acid exhibits the physicochemical properties of an extracellular space marker, though this compound was shown to leak slowly into myocardial cells. Favorable attributes of this indicator are its low molecular weight, high diffusibility in interstitial fluid, low binding affinity to macromolecules, and high transcapillary (1.8 X 10(-5) cm/s) as well as low transplasmalemmal penetration rate (3.7 X 10(-8) cm/s). Therefore, this nonradioactive marker can be applied in a safe and simple fashion, and without untoward side effects in experimental animals as well as in patients.

Determination of the extracellular space with nonradioactive Co3+EDTA and simultaneous estimation of Na, K, Ca, and Mg contents in isolated guinea-pig heart preparations by atomic absorption spectrometry.

A nonradioactive assay for the estimation of the size of the extracellular space (ECS) was developed utilizing Co3+EDTA as ECS marker. Co was measured by atomic absorption spectrophotometry. In a Langendorff preparation the compound was pharmacologically inert at a concentration of 3 X 10(-5) mol/l. The size of the ECS was independent of the ECS marker concentration indicating the lack of tissue adsorption. The filling of the ECS is almost completed after 3 min of perfusion. An insignificant slope of the late phase of the uptake curves in guinea-pig Langendorff heart preparations and isolated atria indicated no or very slow uptake into deep compartments. A sufficiently good estimate of the size of the ECS can be found after 15 min of equilibration. Simultaneous measurements of the size of the ECS and of ions (Na, K, Ca, Mg) can be performed within the same sample. Calculations of cellular Na, K, Mg, and Ca concentrations are in reasonable agreement with literature data.

Influence of Saponins on Gut Permeability and Active Nutrient Transport in Vitro

The influence of four saponins, three triterpenoid glycosides and one steroidal amine glycoside, upon intestinal transport was investigated in vitro. In the presence of Gypsophylla saponin, carrier-mediated galactose transport was inhibited, although the uptake of the passively transported L-isomer of glucose increased. The uptake of the extracellular space marker, polyethylene glycol 4000, was also higher, indicating that the saponin inhibited active transport by increasing the general permeability of the enterocytes. Gypsophylla saponin, in contact only with the mucosal surface of everted jejunal sacs, induced a rapid decline in glucose-stimulated transmural potential difference. The rate of decline increased as the saponin concentration was raised over the approximate range of 0.3 to 8 mM. Saponaria saponin and α-tomatine also reduced transmural potential difference, but soya saponins were much less effective. The results indicate that some saponins readily increase the permeability of the small intestinal mucosal cells, thereby inhibiting active nutrient transport, and facilitating the uptake of materials to which the gut would normally be impermeable.

Effect of CO 2 on a brain extracellular space marker and evidence of its neuronal modulation

Increases in inspired CO 2 consistently altered the local concentration of the brain extracellular space marker α-naphthalene sulfonate (α-NS) as measured with ion-selective micropipettes in the rat thalamus. Stereotaxic injection of lidocaine in the region of the loccus coeruleus attenuated this effect of CO 2, and amittriptyline, a tricyclic anti-depressant and amine reuptake inhibitor, potentiated the effect. These results suggest that metabolic demand, as mimicked here by the addition of CO 2, alters the fluid environment of the brain and central noradrenergic mechanisms may modulate this response.

Alpha-adrenoceptor stimulation, lysophosphoglycerides, and lipid peroxidation in reoxygenation induced calcium uptake in rabbit myocardium.

Calcium uptake on reoxygenation of hypoxic cardiac muscle is well documented. Alpha-adrenoceptor stimulation by released catecholamines, lysophosphoglycerides and lipid peroxidation have all been suggested as mediators of this effect. We have measured the uptake of Ca2+ on reoxygenation in the isolated arterially perfused interventricular septum of the rabbit heart. The alpha agonist phenylephrine (1 mumol X litre-1) did not alter calcium uptake, and the presence of either prazosin (1 mumol X litre-1) or phentolamine (10 mumol X litre-1) did not alter the reoxygenation induced Ca2+ uptake. Lysophosphatidylcholine caused an increase in Ca2+ uptake above 8 mumol X litre-1 but also produced a simultaneous increase in the distribution volume of 51Cr-EDTA, an extracellular space marker, indicating loss of membrane integrity. Hydrogen peroxide and cumene hydroperoxide both caused an increased Ca2+ uptake, but no disruption of the cell membrane; the effect on Ca2+ uptake could be inhibited by Ni2+ ions. Alpha adrenergic stimulation and lysophosphoglycerides do not appear to be key to Ca2+ uptake on reoxygenation, but lipid peroxidation of the sarcolemma is a possible mechanism.

Influence of Age of Culture and Light Intensity on Solute Concentrations in TwoDunaliella Strains

Two strains of Dunaliella, one halotolerant and one halophilic, were grown in batch culture at NaCl concentrations varying from 500 mol m~ 3 to 3000 mol m ~ 3. Measurements were made of the following solutes: glycerol, Na+, K+, Mg2+, CP, phosphate in the cells of logarithmic and of stationary-phase cultures. The method used was to separate the cells from the bulk of the medium by differential density centrifugation. Solute concentrations were calculated using Blue Dextran as a marker for extracellular space. It was found that in log-phase cells, glycerol accounted for one-half to two-thirds of the total cell solute concentration, the remainder being largely accounted for by Na+ and CP. In the stationary phase glycerol fell and Na+, plus CP, rose. Light intensity was found to affect cell volume and solute content. The means whereby solute concentrations are controlled is discussed.

A stopped flow capillary perfusion method to evaluate contraluminal transport parameters of methylsuccinate from interstitium into renal proximal tubular cells.

In order to study the transport of dicarboxylic acids through the contraluminal cell membrane of proximal tubular cells, 3H- methylsuccinate has been synthetized by catalytic hydration of methylfumarate . As the chromatography of radioactive material excreted in the urine after i.v. injection of 3H- methylsuccinate shows, no metabolite is detectable during the first 3 min. After 10 min, less than 10% of the excreted radiolabel is metabolized. To measure the contraluminal influx of 3H- methylsuccinate from the interstitium into cortical tubular cells, the renal vessels were clamped so that the proximal tubular lumina collapsed. Then Ringer solution was injected into the blood capillaries. It contained different concentrations of 3H- methylsuccinate and 14C-inulin as extracellular space marker. After contact times between 1 and 10 s, this fluid was withdrawn from the capillaries and the disappearance of 3H- methylsuccinate relative to 14C-inulin was measured. The morphological compartments in the outer cortex of the clamped glutaraldehyde-fixed kidney were evaluated by a stereological method. For proximal tubular cells a ratio of extracellular water space to intracellular space of 1:3.1 and a ratio extracellular water space to free cell water space of 1:2 was found. It was tested whether the experimental disappearance curves with 4 different starting concentrations of 3H- methylsuccinate fit with the data from four model calculations. It was found that the data and the conditions of transport are consistent with the predictions of a facilitated diffusion model.(ABSTRACT TRUNCATED AT 250 WORDS)

Creatine phosphate: An additive myocardial protective and antiarrhythmic agent in cardioplegia

The potential for enhancing myocardial protection by adding high-energy phosphates to cardioplegic solutions was investigated in a rat heart model of cardiopulmonary bypass and ischemic arrest. Creatine phosphate (CP) was evaluated as an additive to the St. Thomas' Hospital cardioplegic solution. Dose-response studies (CP 0 to 50 mmol/L) revealed 10.0 mmol/L as the optimal concentration which improved recovery of aortic flow and cardiac output after a 40 minute period of normothermic (37 degrees C) ischemic arrest from 21.2% +/- 5.4% and 32.8% +/- 4.6% in the CP-free control group to 82.5% +/- 3.7% and 82.6% +/- 4.2% (p less than 0.001), respectively. Creatine kinase (CK) leakage was reduced by 68.7% (p less than 0.001) in the CP group. With hypothermic (20 degrees C) ischemia (240 minutes) and multidose (every 30 minutes) cardioplegia, recoveries of aortic flow and cardiac output were improved from 33.1% +/- 8.4% and 42.2% +/- 7.7% in the CP-free control group to 77.9% +/- 4.2% and 79.6% +/- 4.3% (p less than 0.001), respectively, in the drug group. In addition to improving function and decreasing CK release, CP reduced reperfusion arrhythmias, significantly decreasing the time between cross-clamp removal and return of regular rhythm and also completely obviating the need for electrical defibrillation. 51Chromium-ethylenediaminetetraacetic acid (51Cr-EDTA), an extracellular space marker, was used to study the disappearance of CP from the cardioplegic solution during its stasis in the heart. Upon reperfusion, two thirds of the infused dose appeared unchanged in the coronary effluent; the remainder was either degraded or accumulated by the myocardium. Despite its alleged inability to enter the myocardial cell, exogenous CP exerts potent protective and antiarrhythmic effects when added to the St. Thomas' Hospital cardioplegic solution. Although the mechanism of action remains to be elucidated, it may involve binding or uptake of the drug.

Comparative analysis of leucine transport in temperate fish liver in vivo

1. 1. The uptake of [ 14C]leucine in toadfish ( Opsanus tau) liver in vivo at 10°C has been studied by a single pulse injection technique. Transport parameters were determined on the basis of the distribution of the amino acid and of [ 3H]inulin, used as a marker for extracellular space, in liver free and protein-bound fractions and in venous blood draining from the liver. 2. 2. Saturation analysis by the Cornish-Bowden method yielded a maximal uptake of 0.26 μmole at 10°C, about half that observed at 21°C. The dose of half-maximal uptake was 0.9 μmole, which was similar on a concentration basis to that at 21°C when circulation rate and dilution with blood are taken into account. 3. 3. Isoleucine and phenylalanine competed with leucine uptake at 10°C as at 21°C; additional competitors at 10°C included histidine, methionine and valine. Fish acclimated to 10°C for 2 weeks or more showed a restoration in maximal leucine uptake and disappearance of histidine inhibition. Methionine inhibition was retained. 4. 4. Three transport systems in this species are discussed: 20-20, operating in 20°C-acclimated fish at 20°C; 20-10, in 20°C-acclimated fish at 10°C; and 10-10, in 10°C-acclimated fish at 10°C. The properties of these systems are compared with the 0-0 system of Antarctic fish and with transport systems of mammalian cells. The latter are similar to our non-acclimated system, 20-10, suggesting that the mammalian cell may not be at a state of optimal temperature adaptation.

D-[1-14C]Mannitol and [U-14C]sucrose as extracellular space markers for human spermatozoa and the uptake of 2-deoxyglucose

[U-14C]Sucrose and D-[1-14C]mannitol were used to determine the tritiated water space of human spermatozoa to validate these compounds as markers for the extracellular space. Calculations based on 0.03 mM-[U-14C]sucrose gave a negative water space. The water space estimated with 0.03 mM-D[1-14C]mannitol was unstable but a stable result was obtained with 0.3 mM-D-[1-14C]mannitol in incubations up to 2 h. The mean water space was 2.21 +/- 0.106 microliters/10(8) spermatozoa (mean +/- s.e.m. for 6 batches of pooled semen). The water space was decreased or abolished by Triton X-100, cold shock, sonication or hypotonic treatment. The water space responded to changes in the osmolarity of the medium by increasing in dilute media. It is concluded that mannitol is an effective extracellular marker for human spermatozoa if concentrations greater than or equal to 0.3 mM are used. When the kinetics of the uptake of 2-deoxyglucose by the spermatozoa were studied by using mannitol as an extracellular marker, the transport was saturable and was inhibited by cytochalasin B. The Km was 1.6 +/- 0.33 mM and the Vmax was 4.2 +/- 0.52 nmol/10(8) spermatozoa/10 sec (mean +/- s.e.m., n = 4).

Relation between the increase in the diffusional permeability of the blood-central nervous system barrier and other changes during the development of experimental allergic encephalomyelitis in the lewis rat

The reduction in the effectiveness of the blood-brain and blood-spinal cord barriers, previously seen in rats at the height of the acute episode of experimental allergic encephalomyelitis, has now been measured at various stages in the development of the disease up to 60 days after inoculation with guinea pig spinal cord in complete Freund's adjuvants. The marker of extracellular space, radioactively labelled mannitol, only crosses the blood-central nervous system barriers very slowly by passive diffusion in normal rats. An abnormal penetration of this marker into the central nervous system began to develop during the second week after inoculation, appearing first in the lower spinal cord, where it also reaches the highest level during the acute phase of the attack. The leak begins before either the clinical signs become evident or cuffing is seen around blood vessels in stained sections. As the clinical signs are disappearing, from about 15 days onwards, the permeability of the barrier returns steadily to its normal low value, starting in the spinal cord, especially the caudal part. The timing of the reduction in the effectiveness of the blood-central nervous system barrier in relation to other clinical and histological changes suggests that it may play a part in the development of the lesion. The relation between the timing of these changes in EAE and that in the development of a new lesion in (exacerbation of) multiple sclerosis is discussed.

Structural Alterations and Changes in the Distribution of Markers in Transected Skeletal Muscle Fibers of the Guinea Pig

The structural alterations produced by transection in striated fibers of cremaster muscle are studied with light and electron microscopes. Diffusion of procion yellow and procion black, which are extracellular space markers, is observed after incubating the transected fibers in the presence or in the absence of Ca<sup>+</sup><sup>+</sup> for 1 h. Cytoplasm at the injured end is always separated from the extracellular space by the sarcolemmal membrane. No outflow of cytoplasmic structures can be seen with light or electron microscopes. The zone of muscle fiber adjacent to the transection is generally dilated. It is occupied by a meshwork of 7-nm-thick filaments. No normal myofibrils are present. The diffusion of procion markers comes to an end within 1 h after the lesion if the fiber is incubated in normal Krebs’ solution. In this period of time they penetrate 250–350 µm inside the fibers. When the transected fibers are incubated in a Ca<sup>+</sup><sup>+</sup> -free solution, procion dyes penetrate about 1 mm. This distance increases in the following 3 h. Diaphragm muscle fibers are used as controls. In these fibers the free penetration of the markers by the injured end is not impeded by Ca<sup>+</sup><sup>+</sup> . These results are in accordance with the calcium-dependent recuperation of the membrane potential 1 h after the transection in cremaster muscle fibers, and with the absence of this recuperation in the diaphragm. It is proposed that the closure of the sarcolemmal membrane creates the diffusion barrier blocking the penetration of markers and ions, allowing the recuperation of membrane potential.

Penetration of horseradish peroxidase into the extravascular channel system of in vitro European eel (Anguilla anguilla) pars distalis, with comments on the role of the nongranulated cells

Horseradish peroxidase (HPO) was used as an extracellular space marker to investigate the characteristics of the extravascular network in in vitro cultured hemipituitaries of Anguilla anguilla. HPO penetrated large extravascular channels in both the rostral and proximal pars distalis. In the rostral pars distalis the channels were associated with the base of the columnar prolactin cells and in the basal lamina between the corticotropes and anterior neurohypophysis in the dorsal region. In the proximal pars distalis the channels separated the cords of granulated cells. HPO also penetrated into fine extracellular spaces, which appeared to be continuous with the extravascular channels, in both rostral and proximal pars distalis. HPO penetrated only those extracellular spaces associated with the nongranulated (NG) cells, suggesting a related function between the movement of large molecules in the extravascular network and NG cell function. However, in in vitro situations which alter pars distalis cell activity (altered osmotic pressure of the incubation medium or incubation in the presence of metabolic inhibitors such as dinitrophenol or KCN) penetration of HPO did not appear to be affected, suggesting that the HPO penetration was passive, at least in the in vitro conditions used here.

Synthesis and use of radio cobaltic EDTA as an extracellular marker in rabbit heart.

A new gamma-labeled marker for extracellular space is the cobaltic form of 58Co-ethylenediaminetetraacetic acid (58Co-EDTA). The cobaltic ion has a much higher affinity for EDTA than the cobaltous ion; it is prepared as a potassium salt, K+(58Co3+-EDTA4-), and is apparently biologically inert. Testing by equilibration in intact rabbits and comparing the myocardial content with that of [14C]sucrose give values of the volume of distribution in the myocardium of 0.294 +/- 0.052 ml/g for 58Co-EDTA and 0.303 +/- 0.051 ml/g for [14C]sucrose (SD, n = 130, for two hearts), with the ratios of 58Co-EDTA/sucrose averaging 0.973 +/- 0.043 (n = 130). The average value of the extracellular fluid measured in isolated rabbit interventricular septum using Co-EDTA was 0.51 +/- 0.05 ml/g (SD, n = 16) and 0.46 +/- 0.04 ml/g using [14C]sucrose as an extracellular fluid space (ECF) marker. Flushing with a high concentration of nontracer Co-EDTA does not reveal any release from binding sites. The gamma-energy (811 KeV), long half-life (71.4 days), stability, and lack of binding to tissue components make 58Co-EDTA a useful marker for ECF.