Total Water Space Research Articles

Rapid Method To Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in Organic Cation Transporter 1-Transfected Human Embryonic Kidney 293 Cells.

Because of the importance of intracellular unbound drug concentrations in the prediction of in vivo concentrations that are determinants of drug efficacy and toxicity, a number of assays have been developed to assess in vitro unbound concentrations of drugs. Here we present a rapid method to determine the intracellular unbound drug concentrations in cultured cells, and we apply the method along with a mechanistic model to predict concentrations of metformin in subcellular compartments of stably transfected human embryonic kidney 293 (HEK293) cells. Intracellular space (ICS) was calculated by subtracting the [(3)H]-inulin distribution volume (extracellular space, ECS) from the [(14)C]-urea distribution volume (total water space, TWS). Values obtained for intracellular space (mean ± S.E.M.; μl/10(6) cells) of monolayers of HEK cells (HEK-empty vector [EV]) and cells overexpressing human organic cation transporter 1 (HEK-OCT1), 1.21± 0.07 and 1.25±0.06, respectively, were used to determine the intracellular metformin concentrations. After incubation of the cells with 5 µM metformin, the intracellular concentrations were 26.4 ± 7.8 μM and 268 ± 11.0 μM, respectively, in HEK-EV and HEK-OCT1. In addition, intracellular metformin concentrations were lower in high K(+) buffer (140 mM KCl) compared with normal K(+) buffer (5.4 mM KCl) in HEK-OCT1 cells (54.8 ± 3.8 μM and 198.1 ± 11.2 μM, respectively; P < 0.05). Our mechanistic model suggests that, depending on the credible range of assumed physiologic values, the positively charged metformin accumulates to particularly high levels in endoplasmic reticulum and/or mitochondria. This method together with the computational model can be used to determine intracellular unbound concentrations and to predict subcellular accumulation of drugs in other complex systems such as primary cells.

Hemodynamic and Biochemical Alterations in Dogs with Lymphoma after Induction of Chemotherapy

BackgroundDoxorubicin is a common antineoplastic agent with dose‐dependent cardiotoxic adverse effects, and pre‐existing myocardial dysfunction is a contraindication to its use.ObjectivesTo systematically define the hemodynamic and biochemical alterations in dogs undergoing chemotherapy for newly diagnosed lymphoma and assess the reversibility of these alterations with fluid administration.AnimalsTwenty‐one client‐owned dogs with newly diagnosed lymphoma were evaluated 1 week after induction of chemotherapy. Underlying degenerative valve disease was exclusionary. Eighteen healthy age‐ and weight‐matched dogs were used as controls.MethodsPhysical examination, blood pressure by Doppler, echocardiography, and biochemical evaluation (routine serum biochemistry, plasma renin activity and aldosterone concentrations, plasma and urine osmolalities, and urine electrolyte concentrations) were measured in dogs with lymphoma and compared to controls. Dogs with lymphoma received crystalloids IV at 6 mL/kg/h for 24 hours. All variables were reassessed at 4 and 24 hours. Deuterium oxide dilution and bromide dilution were used to determine total body water and extracellular water space, respectively.ResultsBaseline echocardiograms showed significantly smaller chamber dimensions in dogs with lymphoma compared to controls. These changes were reversed by fluid administration. Systolic blood pressure and urine sodium concentration were significantly increased, and bromide dilution space, PCV, urine specific gravity, and urine potassium concentration were significantly decreased compared to controls.Conclusion and Clinical ImportanceEchocardiographic and biochemical abnormalities in dogs with lymphoma appear consistent with volume depletion, and may be the result of systemic hypertension and subsequent pressure natriuresis.

Total water, phosphorus relaxation and inter-atomic organic to inorganic interface are new determinants of trabecular bone integrity.

Bone is the living composite biomaterial having unique structural property. Presently, there is a considerable gap in our understanding of bone structure and composition in the native state, particularly with respect to the trabecular bone, which is metabolically more active than cortical bones, and is readily lost in post-menopausal osteoporosis. We used solid-state nuclear magnetic resonance (NMR) to compare trabecular bone structure and composition in the native state between normal, bone loss and bone restoration conditions in rat. Trabecular osteopenia was induced by lactation as well as prolonged estrogen deficiency (bilateral ovariectomy, Ovx). Ovx rats with established osteopenia were administered with PTH (parathyroid hormone, trabecular restoration group), and restoration was allowed to become comparable to sham Ovx (control) group using bone mineral density (BMD) and µCT determinants. We used a technique combining 1H NMR spectroscopy with 31P and 13C to measure various NMR parameters described below. Our results revealed that trabecular bones had diminished total water content, inorganic phosphorus NMR relaxation time (T1) and space between the collagen and inorganic phosphorus in the osteopenic groups compared to control, and these changes were significantly reversed in the bone restoration group. Remarkably, bound water was decreased in both osteopenic and bone restoration groups compared to control. Total water and T1 correlated strongly with trabecular bone density, volume, thickness, connectivity, spacing and resistance to compression. Bound water did not correlate with any of the microarchitectural and compression parameters. We conclude that total water, T1 and atomic space between the crystal and organic surface are altered in the trabecular bones of osteopenic rats, and PTH reverses these parameters. Furthermore, from these data, it appears that total water and T1 could serve as trabecular surrogates of micro-architecture and compression strength.

Mathematical investigation of two dimensional pattern formation

During the perinatal period the problems of drug disposition are pronounced. The adaptation from the maternal-fetal unit to extrauterine life leads to alterations in drug absorption, distribution, metabolism, and renal elimination. Since both morphology and function of the alimentary tract changes, drugs are absorbed more slowly in the neonate than in older infants. Moreover, the serum protein binding of drugs is reduced in neonates. Consequently a measured plasma concentration may reflect a higher plasma/tissue level in the newborn as compared with adults. If the distribution volume of a drug corresponds to the total body water or extracellular water space, the dosage may be calculated in relation to the individual variations in the body surface area. But this procedure is not applicable for the newborn infant due to impaired metabolic functions of the liver and renal elimination mechanisms. The capacity of drug oxidation and glucuronidation is not fully developed in the neonate. Also glomerular filtration and tubular secretion are reduced during the first weeks of life. Therefore the elimination half-lives of many drugs are considerably prolonged in the newborn compared with that in older infants. As a consequence, individual therapeutic drug monitoring based on pharmacokinetic concepts and assisted by computer programs is becoming increasingly important.

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite, Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and (36)Cl(-) flux measurements was used to investigate the transport of Cl(-) and the Cl(-)-dependent transport of "H(+)-equivalents" in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl(-), resulting in an outward [Cl(-)] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H(+)-equivalents). This was reversed on restoration of extracellular Cl(-). The flux of H(+)-equivalents was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. (36)Cl(-) influx measurements revealed the presence of a Cl(-) uptake mechanism with characteristics similar to those of the Cl(-)-dependent H(+)-equivalent flux. The intracellular concentration of Cl(-) in the parasite was estimated to be approximately 48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl(-) together with H(+)-equivalents, resulting in an intracellular Cl(-) concentration well above that which would occur if Cl(-) ions were distributed passively in accordance with the parasite's large, inwardly negative membrane potential.

Hypoxia effects on cell volume and ion uptake of cerebral microvascular endothelial cells

Increased transport of Na across an intact blood-brain barrier (BBB) contributes to cerebral edema formation in ischemic stroke. Our previous studies have shown that ischemic factors stimulate activity of a luminal BBB Na-K-Cl cotransporter, and we have hypothesized that during ischemia, the cotransporter together with the abluminal Na/K pump mediates increased transport of Na from blood into the brain. However, it is possible that elevated Na-K-Cl cotransporter activity could also cause cell swelling if it outpaces ion efflux pathways. The present study was conducted to evaluate the effects of hypoxia on intracellular volume of BBB cells. Cerebral microvascular endothelial cell (CMEC) monolayers were exposed to varying levels of hypoxia for 1 to 5 h in an O(2)-controlled glove box, and cell volume was assessed using 3-O-methyl-D-[(3)H]glucose and [(14)C]sucrose as markers of total and extracellular water space, respectively. Cells exposed to either 7.5%, 3%, or 1% O(2) showed gradual increases in volume (compared with 19% O(2) normoxic controls) that became significant after 3 or more hours. By ion chromatography methods, we also found that a 30-min exposure to 7.5% O(2) caused an increase in bumetanide-sensitive net Na uptake by the cells without increasing cell Na content. CMEC Na content was significantly increased, however, following 3 or more hours of exposure to 7.5% O(2). These findings are consistent with the hypothesis that during cerebral ischemia, the BBB Na-K-Cl cotransporter is stimulated to mediate transendothelial uptake of Na into the brain and that increased cotransporter activity also contributes to gradual swelling of the cells.

Prediction of in vivo tissue distribution from in vitro data 1. Experiments with markers of aqueous spaces.

The aim of this study was to evaluate the ability of an in vitro method of tissue distribution to accurately predict total water and extracellular aqueous spaces using marker compounds urea and inulin. Slices (50-200 mg) of all the major tissues in the rat were incubated with Hanks/HEPES pH7.4 buffer containing 14C-urea and 3H-inulin for 2 h at 37 degrees C. Tissue weight was noted before and after incubation and the tissue-to-buffer ratios determined. 14C-Urea Kp estimates were generally greater than total tissue water due to tissue swelling, which varied widely among the tissues, up to 41% in muscle. In most cases, Kp values were much closer to in vivo values after correcting for the 14C-urea in the imbibed media (Kpcorr). The method was able to distinguish between 14C-urea and 3H-inulin Kp values and indicated that inulin occupied a smaller space than urea, which for the majority of tissues corresponded to the extracellular space. The Kp(corr) values for 14C-urea and Kp for 3H-inulin were consistent with total tissue water and extracellular space for the majority of tissues studied, indicating their suitability as marker compounds for checking the viability of this in vitro method for estimating tissue distribution.

Estimations of intra- and extracellular volume and pH by 31P magnetic resonance spectroscopy: effect of therapy on RIF-1 tumours.

Quantification of metabolite or drug concentrations in living tissues requires determination of intra- and extracellular volumes. This study demonstrates how this can be achieved non-invasively by 31P magnetic resonance spectroscopy (MRS) employing dimethyl methylphosphonate (DMMP) as a marker of total water space, 3-aminopropylphosphonate (3-APP) as a marker of extracellular space and P and 3-APP as markers of intracellular pH (pH) and extracellular pH (pHe) respectively. The MRS measurements of the tumour volumes were validated by classic radiolabelling methods using 3H2O and [14C]inulin as markers of total and extracellular space respectively. The extracellular volume fraction measured by radiolabelling of RIF-1 tumours was 23 +/- 0.83% (mean +/- s.e.m. n = 9), not significantly different (P > 0.1) from that found by MRS (27 +/- 2.9%, n = 9, London, and 35 +/- 6.7, n = 14, Baltimore). In untreated RIF-1 tumours, pH was about 0.2 units higher than pHe (P < 0.01). 5-Fluorouracil (5FU) treatment (165 mg kg(-1)) caused no significant changes in either pHe or per cent extracellular volume. However significant increases in pH, 48 h after treatment (P < 0.01) correlated with decreased tumour size and improved bioenergetic status [NTP/inorganic phosphate (Pi) ratio]. This study shows the feasibility of an MR method (verified by a 'gold standard') for studying the effects of drug treatment on intra- and extracellular spaces and pH in solid tumours in vivo.

Estimation of specific hepatic arterial water space.

The aim of this study was to estimate the specific arterial water space and associated blood flow using statistical moments of the frequency versus time outflow profile, with a model with specific spaces for hepatic arterial (HA) and portal venous (PV) flows in parallel with a common space. Studies were performed in the in situ dual-perfused rat liver (n = 6-10), using Krebs-bicarbonate buffer with constant PV flow (12 ml/min) and various HA flow rates (3-6 ml/min). An impulse input-output technique was employed, varying the route of input, using [14C]urea as the reference indicator. Regardless of flow conditions, the frequency outflow profile after HA input was flatter and broader and the mean transit time longer than after PV input. Excellent recovery of marker was obtained in all cases. Applying the above model, the specific arterial space was estimated to be 9.7 +/- 2.3 of total water space and receives approximately 17% of the HA flow, with the remainder mixing with portal blood in the common space. The estimated total water content of liver (0.67-0.72 ml/g liver) agrees well with that determined by desiccation (0.72 +/- 0.01 ml/g liver).

Response of cell volume in Mytilus gill to acute salinity change.

The response of gill cell volume in Mytilus californianus and Mytilus trossolus (=edulis) to acute changes in salinity was assessed using three independent indicators: optical measurement of lateral cell height, measurement of intracellular water content using radiolabeled tracers and measurement of the contents of the major osmolytes of the gills. Optical measurements indicated significant variation in the response of individual lateral cells of M. californianus to acute low-salinity shock. Lateral cell height increased by approximately 20% shortly after abrupt exposure to 60% artificial sea water (ASW). Following this initial swelling, we estimate that a substantial regulatory volume decrease (RVD) was present in 25% of the trials. More commonly, however, an RVD was either absent or minimal: cell height remained elevated for at least 1 h, then returned to the control height when gills were re-exposed to 100% ASW. Changes in the combined water space of all cells in the gill, measured as the difference between total water space and extracellular space ([14C]polyethylene glycol space), indicated that cell volume regulation in the gill as an organ was also absent or minimal. Cell water space was 2.16 ml g-1 dry mass in isolated gills of M. californianus acclimated to 100% sea water in the laboratory and increased to 2.83 ml g-1 dry mass after a 6 min exposure to 60% ASW. Cell water space was still 2.81 ml g-1 dry mass after 1 h in 60% ASW and returned to 2.06 ml g-1 dry mass upon re-exposure to 100% ASW. Consistent with these observations, the gill contents of the principal cytoplasmic osmolytes (taurine, betaine and K+) were unchanged (approximately 450, 250 and 230 mu mol g-1 dry mass, respectively) following exposure of gills from 100% ASW-acclimated mussels to 60% ASW. A decrease in cell water space to 2.66 ml g-1 dry mass after 4 weeks of acclimation to 60% ASW corresponded with a 37% decrease in betaine content; taurine and K+ contents were unchanged. The changes in water space and solute content of gills from freshly collected M. californianus and M. trossolus were also consistent with the absence of volume regulation; cell water space remained elevated for at least 1 h after low-salinity exposure, and solute contents were unchanged after this period. We calculated the potential energetic cost of cell volume regulation for mussels exposed to 12 h of sinusoidal fluctuations between 100% and 50% sea water; solute uptake for full volume regulation in all tissues would cost a minimum of approximately 30% of the standard metabolic rate during the period of salinity increase. The routine absence of substantial cell volume regulation in Mytilus gill may reflect the potentially high energetic cost of volume regulation in the face of the large and frequent salinity fluctuations that are regularly encountered by estuarine bivalves.

Stress‐dependent redistribution of abscisic acid (ABA) in Hordeum vulgare L leaves: the role of epidermal ABA metabolism, tonoplastic transport and the cuticle

ABSTRACTWhen 14C‐labelled abscisic acid ([14C]ABA) was supplied to isolated protoplasts of the barley leaf at pH 6, initial rates of metabolism were about five times higher in epidermal cell protoplasts than in mesophyll cell protoplasts if equal cytosolic volumes were considered. In spite of the fact that epidermal cells make up only about 35% of the total water space in barley leaves, and despite the small cytosolic volume of these cells, in intact leaves all epidermal cells would thus metabolize half as much ABA per unit time as the mesophyll cells (0–27 and 0–51 mmol h−1 m−3 leaf water). Therefore, under these conditions epidermal cells seem to be a stronger sink than mesophyll cells for ABA that arrives via the transpiration stream. However, at an apoplastic pH of 7–25, which occurs in stressed leaves, the proportion of total metabolized ABA would be much smaller in epidermal than in mesophyll cells (0–029 and 0–204 mmolh−l m−3 leaf water). Our results indicate that under conditions of slightly alkaline apoplastic pH the epidermis may serve as the main source for fast stress‐dependent ABA redistribution into the guard cell apoplast. This is partly the result of ABA transport across the epidermal tonoplast, which is dependent on the apoplastic pH and possibly on the cytosolic calcium concentration. The cuticle seems to be of no particular importance in stress‐induced apoplastic ABA shifts and cannot be regarded as a significant sink for high ABA concentrations under stress.

Pulmonary uptake of bupivacaine in isolated perfused rat lung.

The ability of rat lung to remove the local anaesthetic drug bupivacaine from the blood was studied in isolated organs which were perfused either in an open (single-pass mode) or in a closed system (recirculating medium). Isolated perfused rat lungs exhibited a very low capacity to metabolize bupivacaine within 3 h during which the drug circulated continuously through the organ. The clearance values differed only by 0.2 ml/min from the control parameters in sham perfusions. The calculated extraction ratio was 0.2% and the elimination half-life was about 210 min. The volume of distribution of bupivacaine was 133 ml which remarkably surmounted the reference values obtained for sham perfusions. The distribution of bupivacaine into the pulmonary tissue was investigated applying the multiple indicator dilution technique to isolated lungs perfused in the single-pass mode. The mean elimination time of model compounds for distribution into the intravascular space, 14C-insulin, and the total water space, 3H-water, were 68 and 75 s at a flow rate of 6 ml/min. The volume of distribution was 5.9 ml for inulin and 6.5 ml for water. The mean transit time for concomitantly injected bupivacaine was 221 s and the volume of distribution was 14.4 ml. The respective parameters of sham perfusions performed without an isolated organ were substantially lower, i.e. mean elimination time 50, 50 and 61 s and distribution volume 4.9, 5.0 and 6.1 ml for inulin, water and bupivacaine.2+ f1p4

Role of the Hepatic Artery in the Metabolism of Phenacetin and Acetaminophen: An Intravital Microscopic and Multiple–Indicator Dilution Study in Perfused Rat Liver

We studied the pattern of intermixing of the hepatic arterial and portal venous flows in a perfused rat liver preparation under constant flow (12 ml/min) with intravital epifluorescent microscopy; changes in the steady state extraction ratio of carbon 14-labeled phenacetin and tritiated acetaminophen, probes metabolized primarily in perivenous and periportal regions of the rat liver, respectively; and the spaces accessed by noneliminated reference indicators introduced as a bolus into the hepatic artery and portal vein at different hepatic arterial/portal venous flow regimens of 0:12, 2:10 and 4:8. The sinusoidal velocities for the hepatic arterial- and portal venous (hepatic arterial/portal venous flow at 4:8)-infused fluorescein isothiocyanate-erythrocytes (100 microliters/min) were 327 +/- 78 and 301 +/- 63 microns/sec, respectively, and the velocity for the solely portal venous-perfused liver (12 ml/min) was 347 +/- 74 microns/sec; the flow-weighted sinusoidal velocity was highly correlated to the sinusoidal volume for the dually perfused rat liver. Small but significant decreases in the extraction ratio of [14C]phenacetin (from 0.989 to 0.984 and 0.980) and tritiated acetaminophen (from 0.631 to 0.607 to 0.563), delivered simultaneously into the hepatic artery and portal vein, were observed with an increment of hepatic arterial flow within the same liver preparation; oxygen consumption rate also fell slightly, in parallel fashion. When a multiple-indicator dilution dose containing chromium 51-labeled RBCs, iodine 125-labeled albumin and tritiated water or [14C]urea was injected into the hepatic artery (which accesses both the peribiliary capillary plexus [nonsinusoidal] and the sinusoidal bed) and portal vein (which enters only the sinusoids) at 10-min intervals within each steady state, the blood volume, total albumin space, albumin Disse space, total water and parenchymal cellular water spaces were unchanged after portal venous injection for all hepatic arterial/portal venous flow ratios, suggesting that the arterial flow is ineffective in perturbing average sinusoidal flow dynamics. However, slightly larger total water spaces were obtained with hepatic arterial injection. This excess water space was almost completely accounted for by the "nonsinusoidal" extravascular space associated with the peribiliary capillary plexus; it averaged 0.03 ml/gm and was independent of flow. The anomaly, a reduced flow-weighted sinusoidal velocity for the dually perfused liver, an unchanged diameter of the terminal hepatic venule (32 microns) among the hepatic arterial/portal venous flow ratios and the reduction in the extraction ratio of the drug probes and oxygen consumption rates suggest that some of the arterial flow must have entered the sinusoids somewhat downstream.

Intracellular and extracellular spaces and the direct quantification of molar intracellular concentrations of phosphorus metabolites in the isolated rat heart using 31P NMR spectroscopy and phosphonate markers

To quantify metabolite and cation concentrations using NMR spectroscopy, the volumes of intracellular and extracellular spaces must be known. We describe a simple 31P NMR spectroscopic method that employs dimethyl methylphosphonate (DMMP) as a marker of total water space and phenylphosphonate (PPA) as a marker of extracellular space to determine intracellular and extracellular space volumes in the isolated, perfused rat heart. In vivo and in vitro radiolabel studies were used to verify this method. The difference between the total and extracellular water spaces, determined as milliliters/heart, gave the intracellular volume and allowed direct calculation of myocardial creatine phosphate, ATP, and inorganic phosphate concentrations, which were 13.4 mM, 10.1 mM, and 3.4 mM, respectively, for the glucose-perfused rat heart. The extracellular volume decreased by 84% in hearts subjected to 28 min total, global ischemia and increased by 15% during reperfusion. The method described allows the determination of intracellular energy metabolite concentrations in perfused rat heart directly from a single, fully relaxed 31P NMR spectrum.

31P Magnetic Resonance Spectroscopy Demonstrates Expansion of the Extracellular Space in the Skeletal Muscle of Starved Rats

Starvation significantly alters the distribution of body water. To study the effects of starvation on cellular energetics and water distribution in skeletal muscle, a novel 31P magnetic resonance technique (31P MRS) was developed to measure water compartments. After 31P MRS-visible water space markers which distribute in total body water (dimethyl methylphosphonate, DMMP) and extracellular water (phenylphosphonate, PPA) were infused intravenously, 31P MRS spectra were obtained from the gastrocnemius muscle of male virus-free Wistar rats at baseline and after starvation or ad libitum feeding for 4 days. Muscle water spaces were also measured using the chloride method and Nernst's equation. Muscle water contents as determined by drying were equivalent in the two groups. In vivo measurements of changes in DMMP relative to all of the MRS visible phosphates also demonstrated that the total water space was similar in control and starved rats. However, starvation significantly increased the ratio of PPA/DMMP (0.67 ± 0.05 vs 0.87 ± 0.04, Control vs Starvation; P < 0.001), and therefore the ratio of extracellular water to total water in the gastrocnemius. Furthermore, because muscle water contents were comparable between the groups, this expansion of the extracellular space was accompanied by contraction of the intracellular compartment in starved animals. Equivalent changes were detected in vitro using the chloride method. Lastly, phosphocreatine/ATP ratios, which measured changes in high-energy phosphate stores, decreased after starvation (4.09 ± 0.06 vs 3.61 ± 0.06; P < 0.001) and were inversely related to changes in PPA/DMMP (r = -0.61; P < 0.001).We conclude that starvation alters the distribution of water within skeletal muscle and these changes are related to the depletion of energy stores. These phenomena can be studied simultaneously in a noninvasive fashion using in vivo31P MRS and MRS-visible water space markers.

Physiological pharmacokinetics of solutes in the isolated perfused rat hindlimb: Characterization of the physiology with changing perfusate flow, protein content, and temperature using statistical moment analysis

Distribution of Evans Blue (EB), sucrose, and water into the isolated perfused rat hindlimb was studied under various conditions using the multiple indicator dilution (MID) technique. Statistical moment analysis of the outflow profiles for the EB, sucrose, and water were used to define the vascular, extravascular, and total water spaces, respectively. The varied perfusion conditions included albumin content (2, 4.7, and 7%), temperature (25, 37, and 42 C), perfusate flow rate (2, 4, 8, and 12 ml/min) and the presence/absence of red blood cells. The range of studies undertaken were chosen to represent the variety of conditions used in the preparation of both isolated animal and human limbs, the latter being particularly important in cytotoxic therapy for recurrent malignant melanoma. The distribution volumes of EB, sucrose, and water were dependent on the flow rate and the albumin content of perfusate. The normalized variances (CV2) of the markers were of the following order: sucrose (2.18) > water (1.58) > EB (0.68), indicating that some disequilibrium occurs during the capillary exchange of water and sucrose. It is suggested that a Krebs-Henseleit buffer containing 2% BSA is a suitable perfusate for most studies of the isolated rat hindlimb perfusion. The effect of albumin concentration manifests itself only at higher flows.

Multinuclear NMR studies of the Langendorff perfused rat heart

The quantitation of intracellular sodium ion concentration [Na+]in perfused organs using NMR spectroscopy requires a knowledge of the extent of visibility of the 23Na resonance and of the intracellular volume of the organ. We have used a multinuclear NMR approach, in combination with the extracellular shift reagent dysprosium (III) tripolyphosphate, to determine the NMR visibility of intra- and extracellular 23Na and 35Cl ions, intracellular volume, and [Na+]in in the isolated Langendorff perfused rat heart. Based on a comparison of the extracellular volumes calculated using 2H and 23Na, 35Cl, or 59Co NMR of the perfused heart we conclude that resonances of extracellular sodium and chloride ions (including ions in interstitial spaces) are fully visible, contrary to assumptions in the literature. Furthermore, prolonged hypoxia or ischemia caused a dramatic increase in intracellular Na+ and [Na+] in rose to approach that in the external medium indicating full visibility of the intracellular 23Na resonance. Resonance intensities of intra- and extracellular 23Na ions, along with a knowledge of the extracellular space as a fraction of the total organ water space, yielded an average [Na+] in of about 10 mM (10 +/- 1.5 mM) for the rat heart at 37 degrees C. Double-quantum filtered 23Na NMR of the perfused rat heart in the absence and presence of paramagnetic reagents revealed, contrary to assumptions in the literature, that both intra- and extracellular sodium ions contribute to the detected signal.

&lt;sup&gt;23&lt;/sup&gt;Na, &lt;sup&gt;19&lt;/sup&gt;F, &lt;sup&gt;35&lt;/sup&gt;Cl and &lt;sup&gt;31&lt;/sup&gt;P Multinuclear Nuclear Magnetic Resonance Studies of Perfused Rat Kidney

The concentration of intracellular sodium [Na+]i has been measured in the perfused rat kidney using 23Na nuclear magnetic resonance (NMR) in combination with the extracellular shift reagent Dy(PPPi)7-(2). The data show 100% visibility of Na+ in interstitial spaces. A measurement of the resonance intensities of intra- and extracellular 23Na ions along with a knowledge of the extracellular space as a fraction of the total kidney water space yielded an average [Na+]i of 27 +/- 2 mM for the kidney at 37 degrees C. After prolonged ischemia [Na+]i rose to approach that in the external medium. In the absence of 5% albumin in the perfusion medium, the linewidth of the 35Cl resonance of an adult kidney (45 Hz) was about twofold larger than that of the medium alone (25 Hz). In contrast, the linewidth of 35Cl resonance of an adult kidney perfused with an albumin-containing medium (82 Hz) was only about 27% of that from the medium alone (300 Hz). We interpret this effect to be due to compartmentation of albumin in the extracellular space such that the interstitial space is not freely accessible to albumin. However, for a developing, immature kidney from a growing animal, perfused with an albumin-containing medium, the linewidth of the 35Cl resonance (233 Hz) was only slightly less than that of the medium alone (300 Hz), indicating a much greater albumin permeability of the capillary walls. 19F NMR of a perfused adult kidney, loaded with the membrane-impermeant intracellular calcium indicator 5FBAPTA, yielded a value of 256 nM for [Ca2+]i. Induction of ischemia for 10 min caused the [Ca2+]i to rapidly rise to 660 nM, which could not be fully reversed by reperfusion, suggesting irreversible injury.

Inhibition of monosaccharide transport in the intact rat liver by stevioside

The transport and metabolism of d-glucose and d-fructose in the isolated perfused rat liver and the influence of stevioside and its derivatives were investigated. The transport parameters were measured by the multiple indicator dilution technique. The maximal exchange rate of d-glucose was 700 μmol-min −1.ml −1 and the K m was 38 mM. Stevioside and its derivatives (isosteviol and steviolbioside) inhibited d-glucose and d-fructose transport across the cell membrane. The half-maximal effect at 1 mM d-glucose occurred at 0.8 mM stevioside. The inhibitory action of stevioside was of mixed type. Isosteviol was more potent than stevioside (half-maximal effect at 0.4 mM), whereas steviolbioside was less active (50% inhibition at 2.5 mM). Stevioside was without effect on d-glucose metabolism, except for transient changes in d-glucose release, reflecting changes in the intracellular concentration. DFructose consumption, however, was specifically affected (half-maximal effect at 2.8mM), as well as all parameters depending on D-fructose transformation ( D-glucose production, L-lactate and pyruvate production, and extra oxygen uptake). In livers releasing D-glucose from endogenous glycogen, strong inhibition of transport increased the intracellular to extracellular d-glucose concentration ratio C i C e . The control values of C i C e , representing an average over the total intracellular water space, were always smaller than unity. The latter observation may indicate that D-glucose does not have access to the whole intracellular water space.

Strategies of hemopoietic stress adaptation within the medullary cavity

Gravimetric determination of total bone water space was used as an index of available bone marrow space in mice following various specific stressors, i.e., splenectomy, hypoxia, bone fracture, and estrone-induced osteosclerosis. Data was corrected for bone weight and was reported as specific bone marrow volume (total bone water space/mg dry bone X 100). A direct relationship was observed between specific bone marrow volume and medullary hemopoietic activity induced by stress. Absolute and/or relative marrow space increased following splenectomy, hypoxia, and fracture. Osteosclerotic animals shift most hemopoietic activity from marrow to spleen, and splenectomized osteosclerotic animals become anemic. Both intact and splenectomized hypoxic animals develop increased specific bone marrow volume and successfully compensate for hypoxia with enhanced erythropoiesis. Animals sustaining a fracture callus increase both specific bone marrow volume and hemopoietic activity at the callus without an increase in hemopoietic demand. Increased specific bone marrow volume extends the marrow bone interface, where primitive stem cells accumulate, while expanding marrow stromal space, where stem cells lodge, proliferate and differentiate. Therefore, it would appear that availability of competent marrow space may play an integral part in passively permitting hematopoiesis and in determining hemopoietic reserve capacity. Stem cell migration increases during intensified hemopoietic demand, which also may be related to available marrow space. Mice have a low medullary hemopoietic reserve capacity; subsequently, when available medullary hematopoietic stroma becomes occupied, stem cells are more likely to migrate from the marrow to extra-medullary sites where they mature before entering the circulating pool.