Fluid balance assessment in pediatric hemodialysis patients by using whole-body bioimpedance spectroscopy (WB-BIS).

BACKGROUND: Fluid overload is a major factor in morbidity and mortality in dialysis patients. Whole-body bioimpedance spectroscopy (WB-BIS) is a noninvasive method for assessing fluid status. We hypothesized that fluid status measurement of changes in total body water (TBW), extracellular fluid (ECF), and intracellular fluid (ICF) by WB-BIS would correlate with the weight (Wt) changes before and after hemodialysis (HD) and the amount of ultrafiltration (UF) in pediatric HD patients. We also examined the relationship between the ECF percent of total body water (ECF%) and ECF/ICF ratio with the pre-HD systolic blood pressure percentile (SBP%ile). METHODS: WB-BIS measurements were made both before and after HD on three separate occasions in each patient. Pre- and post-HD Wt, BP, and UF volumes were collected on the day of BIS measurement. RESULTS: At total of 96 measurements were obtained from 16 HD patients. There were 6 females (mean age: 13.2 ± 4.5 yrs). UF correlated with changes in weight, TBW and ECF (p < 0.001) but not with ICF changes (p = 0.345). Pre-HD SBP%ile correlated with ECF%. CONCLUSIONS: Our findings suggest that WB-BIS can be used to monitor the fluid status in pediatric HD patients. The fluid that is removed from the patient during the HD treatment primarily comes from the ECF and not the ICF. Mobilization of fluid from the ICF appears to be delayed. Patients with significantly higher pre-HD ECF% and ECF/ICF ratio had higher pre-HD systolic BP. A higher resolution version of the Graphical abstract is available as Supplementary information.

Health disparities: Intracellular consequences of social determinants of health.

Health disparities exist dependent on socioeconomic status, living conditions, race/ethnicity, diet, and exposures to environmental pollutants. Herein, the various exposures contributing to a person's exposome are collectively considered social determinants of health (SDOH), and the SDOH-exposome impacts health more than health care. This review discusses the extent of evidence of the physiologic consequences of these exposures at the intracellular level. We consider how the SDOH-exposome, which captures how individuals live, work and age, induces cell processes that modulate a conceptual "redox rheostat." Like an electrical resistor, the SDOH-exposome, along with genetic predisposition and age, regulate reductive and oxidative (redox) stress circuits and thereby stimulate inflammation. Regardless of the source of the SDOH-exposome that induces chronic inflammation and immunosenescence, the outcome influences cardiometabolic diseases, cancers, infections, sepsis, neurodegeneration and autoimmune diseases. The endogenous redox rheostat is connected with regulatory molecules such as NAD+/NADH and SIRT1 that drive redox pathways. In addition to these intracellular and mitochondrial processes, we discuss how the SDOH-exposome can influence the balance between metabolism and regulation of immune responsiveness involving the two main molecular drivers of inflammation, the NLRP3 inflammasome and NF-κB induction. Mitochondrial and inflammasome activities play key roles in mediating defenses against pathogens and controlling inflammation before diverse cell death pathways are induced. Specifically, pyroptosis, cell death by inflammation, is intimately associated with common disease outcomes that are influenced by the SDOH-exposome. Redox influences on immunometabolism including protein cysteines and ion fluxes are discussed regarding health outcomes. In summary, this review presents a translational research perspective, with evidence from in vitro and in vivo models as well as clinical and epidemiological studies, to outline the intracellular consequences of the SDOH-exposome that drive health disparities in patients and populations. The relevance of this conceptual and theoretical model considering the SARS-CoV-2 pandemic are highlighted. Finally, the case of asthma is presented as a chronic condition that is modified by adverse SDOH exposures and is manifested through the dysregulation of immune cell redox regulatory processes we highlight in this review.

Quantitative assessment of the spatial crowding heterogeneity in cellular fluids.

Mammalian cells are crowded with macromolecules, supramolecular complexes, and organelles, all of which equip intracellular fluids, e.g., the cytoplasm, with a dynamic and spatially heterogeneous occupied volume fraction. Diffusion in such fluids has been reported to be heterogeneous, i.e., even individual single-particle trajectories feature spatiotemporally varying transport characteristics. Complementing diffusion-based experiments, we have used here an imaging approach to assess the spatial heterogeneity of the nucleoplasm and the cytoplasm in living interphase cells. As a result, we find that the cytoplasm is more crowded and more heterogeneous than the nucleoplasm on several length scales. This phenomenon even persists in dividing cells, where the mitotic spindle region and its periphery form a contiguous fluid but remain nucleoplasmlike and cytoplasmlike, respectively.

Assessment of Anticancer Drug Effects on Pancreatic Cancer Cells under Glucose-Depleted Conditions Using Intracellular and Extracellular Amino Acid Metabolomics.

Previously, we developed a method to evaluate states of cells treated with anticancer drugs via the comprehensive analysis of amino acids, termed amino acid metabolomics. In the present study, we evaluated the effects of the anticancer drugs, gemcitabine hydrochloride and pyrvinium pamoate, on the proliferation of a pancreatic cancer cell line (PANC-1) under hypoglycemic conditions using amino acid metabolomics. Intracellular and extracellular amino acid profiles of PANC-1 were determined by hydrophilic interaction chromatography-tandem mass spectrometry with simple pretreatment. Changes to the drugs' anticancer effects resulting from glucose starvation conditions were presented in score plots obtained from principal component analyses. In particular, the analysis of intracellular amino acids was found to be the superior approach because the results allowed a clearer assessment of the cell state. Further, orthogonal partial least squares discriminant analysis was performed to search for amino acid candidates that discriminate with anticancer drug-treated PANC-1 cells. We identified several amino acids that might be able to distinguish the drug-treated group from the control group. These results might provide a better understanding of the mechanisms underlying cell responses such as drug resistance or austerity. The present study is the first to evaluate the efficacy of anticancer drugs under glucose starvation based on the analysis of the variation of extracellular and intracellular amino acid profiles in vitro.

IL-22 mediates host defense against an intestinal intracellular parasite in the absence of IFN-γ at the cost of Th17-driven immunopathology.

The roles of Th1 and Th17 responses as mediators of host protection and pathology in the intestine are the subjects of intense research. In this study, we investigated a model of intestinal inflammation driven by the intracellular apicomplexan parasite Eimeria falciformis. Although IFN-γ was the predominant cytokine during E. falciformis infection in wild-type mice, it was found to be dispensable for host defense and the development of intestinal inflammation. E. falciformis-infected IFN-γR(-/-) and IFN-γ(-/-) mice developed dramatically exacerbated body weight loss and intestinal pathology, but they surprisingly harbored fewer parasites. This was associated with a striking increase in parasite-specific IL-17A and IL-22 production in the mesenteric lymph nodes and intestine. CD4(+) T cells were found to be the source of IL-17A and IL-22, which drove the recruitment of neutrophils and increased tissue expression of anti-microbial peptides (RegIIIß, RegIIIγ) and matrix metalloproteinase 9. Concurrent neutralization of IL-17A and IL-22 in E. falciformis-infected IFN-γR(-/-) mice resulted in a reduction in infection-induced body weight loss and inflammation and significantly increased parasite shedding. In contrast, neutralization of IL-22 alone was sufficient to increase parasite burden, but it had no effect on body weight loss. Treatment of an E. falciformis-infected intestinal epithelial cell line with IFN-γ, IL-17A, or IL-22 significantly reduced parasite development in vitro. Taken together, to our knowledge these data demonstrate for the first time an antiparasite effect of IL-22 during an intestinal infection, and they suggest that IL-17A and IL-22 have redundant roles in driving intestinal pathology in the absence of IFN-γ signaling.

Simultaneous assessment of uptake and metabolism in rat hepatocytes: a comprehensive mechanistic model.

Kinetic parameters describing hepatic uptake in hepatocytes are frequently estimated without appropriate incorporation of bidirectional passive diffusion, intracellular binding, and metabolism. A mechanistic two-compartment model was developed to describe all of the processes occurring during the in vitro uptake experiments performed in freshly isolated rat hepatocytes plated for 2 h. Uptake of rosuvastatin, pravastatin, pitavastatin, valsartan, bosentan, telmisartan, and repaglinide was investigated over a 0.1 to 300 µM concentration range at 37°C for 2 or 45-90 min; nonspecific binding was taken into account. All concentration-time points were analyzed simultaneously by using a mechanistic two-compartment model describing uptake kinetics [unbound affinity constant (K(m,u)), maximum uptake rate (V(max)), unbound active uptake clearance (CL(active,u))], passive diffusion [unbound passive diffusion clearance (P(diff,u))], and intracellular binding [intracellular unbound fraction (fu(cell))]. When required (telmisartan and repaglinide), the model was extended to account for the metabolism [unbound metabolic clearance (CL(met,u))]. The CL(active,u) ranged 8-fold, reflecting a 11-fold range in uptake K(m,u), with telmisartan and valsartan showing the highest affinity for uptake transporters (K(m,u) <10 µM). Both P(diff,u) and fu(cell) span over two orders of magnitude and reflected the lipophilicity of the drugs in the dataset. An extended incubation time allowed steady state to be reached between media and intracellular compartment concentrations and reduced the error in certain parameter estimates observed with shorter incubation times. Active transport accounted for >70% of total uptake for all drugs investigated and was 4- and 112-fold greater than CL(met,u) for telmisartan and repaglinide, respectively. Modeling of uptake kinetics in conjunction with metabolism improved the precision of the uptake parameter estimates for repaglinide and telmisartan. Recommendations are made for uptake experimental design and modeling strategies.

Resting energy expenditure and body composition in patients with newly detected cancer.

BACKGROUND & AIMS: Elevated resting energy expenditure (REE) may be a major determinant in the development of cancer cachexia. The aim of the study was to evaluate REE and body composition in cancer patients and find out the relationship between energy expenditure and substrate utilization. METHODS: Measured resting energy expenditure (mREE), carbohydrate oxidation (C-O), and fat oxidation (F-O) were measured by indirect calorimetry in 714 cancer patients and 642 controls. Extracellular fluid (ECF), intracellular fluid (ICF), and total water (TW) were measured by bioelectrical impedance appliance; fat mass (FM), fat free mass (FFM), and body cell mass (BCM) were further determined. RESULTS: Compared with the controls, cancer patients showed no significant difference in mREE, but had higher mREE/FFM and mREE/pREE. 46.7% (n=333) of cancer patients were hypermetabolic, 43.5% (n=310) normometabolic, and 9.8% (n=71) hypometabolic; whereas 25.2% (n=162) of control subjects were hypermetabolic, 56.5% (n=363) normometabolic, and 18.3% (n=117) hypometabolic. Cancer patients showed an increase in F-O, ECF, TW/BW and ECF/BW; and a decrease in C-O, npRQ, ICF, ICF/BW. REE was correlated to substrate oxidation rate. Cancer patients exhibited an elevation in FM, FM/BW, FFM, and BCM, and a decrease in FFM/BW. CONCLUSIONS: 1. Cancer patients had elevated REE. Cancer type, pathological stage and duration of disease influenced REE. 2. Aberrations in substrate utilization may contribute to the elevated REE in cancer patients. 3. FM, FFM, and BCM diminished in cancer patients, which may be related to the elevated REE.

Combined PET and microdialysis for in vivo assessment of intracellular drug pharmacokinetics in humans.

UNLABELLED: Because many drugs possess an intracellular site of action, the knowledge of intracellular concentration-time profiles is desirable. In the present study, PET, which measures total (i.e., intracellular, extracellular, and intravascular) concentrations of radiolabeled drugs in tissue, and microdialysis, which determines unbound drug concentrations in the extracellular space fluid of tissue, were combined to describe the intracellular pharmacokinetics of a model compound--that is, the (18)F-labeled antibiotic (18)F-ciprofloxacin--in vivo in humans. METHODS: Ten healthy male volunteers received a mixture of 687 +/- 50 MBq of (18)F-ciprofloxacin and 200 mg of unlabeled ciprofloxacin as an intravenous bolus infusion over 10 min. The pharmacokinetics of ciprofloxacin in skeletal muscle tissue were assessed by means of combined PET and in vivo microdialysis for 5 h after drug administration. A 3-compartment pharmacokinetic model was fitted to the tissue concentration-time profiles of ciprofloxacin measured by PET to estimate the rate constants of ciprofloxacin uptake and transport. RESULTS: In muscle tissue, mean total and extracellular peak concentration (C(max)) values of ciprofloxacin of 1.8 +/- 0.4 microg/mL and 0.7 +/- 0.2 microg/mL were attained at 95 +/- 34 min and 48 +/- 20 min after drug administration, respectively. The extracellular-to-intracellular exchange appeared to be very fast, with an estimated rate constant k(3) of 1.69 +/- 0.25 min(-1). An intracellular-to-extracellular concentration ratio (C(intra)/C(extra)) of 3.2 +/- 0.8 was reached at 110 min after injection and followed by sustained intracellular retention of the antibiotic for the remainder of the experiment. The predicted extracellular concentration-time profiles from the compartmental modeling were in good agreement with the measured microdialysis data. CONCLUSION: The results obtained in the present study were in accordance with previous in vitro data describing cellular ciprofloxacin uptake and retention. The presently used PET/microdialysis combination might be useful during research and development of new drugs, for which knowledge of intracellular concentrations is of interest.

Appreciating the heterogeneity in autoimmune disease: multiparameter assessment of intracellular signaling mechanisms.

Autoimmune disease pathologies are multifactorial with complex interactive networks of cells and chemical messengers that initiate cascades of aberrant cellular activity. Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by systemic inflammation, destruction of the joints, and production of autoantibodies recognizing dozens of putative autoantigens. The presence of autoreactive T cells in individuals leads to pathological autoimmunity by activating additional cellular constituents to mediate inflammation and joint destruction. The etiology of RA is unknown, and knowledge is lacking of the molecular mechanisms underlying the production and subsequent regulation of autoreactive T cells and predicting patient responses to treatments. Biochemical investigations into mechanisms of the disease have relied on animal models that are helpful in dissecting elements of the disease but that are not necessarily reflective of human RA development. The study of multiple activated signaling pathways in complex populations of cells, such as peripheral blood, at the single-cell level has not previously been possible. This article describes how intracellular phosphoepitope staining methodology in conjunction with surface-cell immunophenotyping can be used to deconvolute cellular subsets and allow functional characterization of patient-derived material. Multiparameter flow cytometric analysis allows for small subpopulations-representing different cellular subsets and differentiation or activation states-to be discerned and simultaneously assessed for intracellular biochemical activities. This article also describes how single-cell signal network analysis can be used to stratify patients and may be useful for understanding mechanisms of disease progression, treatment resistance, and development of diagnostic indicators.

GABA uptake into astrocytes is not associated with significant metabolic cost: implications for brain imaging of inhibitory transmission.

Synaptically released glutamate has been identified as a signal coupling excitatory neuronal activity to increased glucose utilization. The proposed mechanism of this coupling involves glutamate uptake into astrocytes resulting in increased intracellular Na+ (Nai+) and activation of the Na+/K+-ATPase. Increased metabolic demand linked to disruption of Nai+ homeostasis activates glucose uptake and glycolysis in astrocytes. Here, we have examined whether a similar neurometabolic coupling could operate for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), also taken up by Na+-dependent transporters into astrocytes. Thus, we have compared the Nai+ response to GABA and glutamate in mouse astrocytes by microspectrofluorimetry. The Nai+ response to GABA consisted of a rapid rise of 4-6 mM followed by a plateau that did not, however, significantly activate the pump. Indeed, the GABA transporter-evoked Na+ influxes are transient in nature, almost totally shutting off within approximately 30 sec of GABA application. The metabolic consequences of the GABA-induced Nai+ response were evaluated by monitoring cellular ATP changes indirectly in single cells and measuring 2-deoxyglucose uptake in astrocyte populations. Both approaches showed that, whereas glutamate induced a robust metabolic response in astrocytes (decreased ATP levels and glucose uptake stimulation), GABA did not cause any measurable metabolic response, consistent with the Nai+ measurements. Results indicate that GABA does not couple inhibitory neuronal activity with glucose utilization, as does glutamate for excitatory neurotransmission, and suggest that GABA-mediated synaptic transmission does not contribute directly to brain imaging signals based on deoxyglucose.

Neuronal activity regulates correlated network properties of spontaneous calcium transients in astrocytes in situ.

Spontaneous neuronal activity is essential to neural development. Until recently, neurons were believed to be the only excitable cells to display spontaneous activity. However, cultured astrocytes and, more recently, astrocytes in situ are now known to exhibit spontaneous Ca2+ transients. Here we used Ca2+ imaging of astrocytes from transgenic mice for the simultaneous monitoring of [Ca2+]i changes in large numbers of astrocytes. We found that spontaneous activity is a common property of most brain astrocytes that is lost in response to a lesion. These spontaneous [Ca2+]i oscillations require extracellular and intracellular Ca2+. Moreover, network analysis revealed that most astrocytes formed correlated networks of dozens of these cells, which were synchronous with both astrocytes and neurons. We found that decreasing spontaneous [Ca2+]i transients in neurons by TTX does not alter the number of active astrocytes, although it impairs their synchronous network activity. Conversely, bicuculline-induced epileptic patterns of [Ca2+]i transients in neurons cause an increase in the number of active astrocytes and in their network synchrony. Furthermore, activation of non-NMDA and NMDA ionotropic glutamate receptors is required to correlate astrocytic networks. These results show that spontaneous activity in astrocytes and neurons is patterned into correlated neuronal/astrocytic networks in which neuronal activity regulates the network properties of astrocytes. This network activity may be essential for neural development and synaptic plasticity.

Assessment of islet beta-cell mass in isolated rat pancreases perfused with D-[(3)H]mannoheptulose.

D-mannoheptulose is apparently transported into cells mainly at the intervention of GLUT-2 and hence was recently proposed as a tool to label preferentially insulin-producing beta-cells in the pancreatic gland. The validity of such a proposal was investigated in the present study conducted in isolated perfused pancreatic glands from control and streptozotocin-induced diabetic rats. After a 30-min equilibration period, D-[(3)H]mannoheptulose (0.1 mM) and [U-(14)C]sucrose (0.5 mM) were infused for 15 min in the presence of 30 mM D-glucose. The pancreatic glands were then perfused for 10 min with a nonradioactive medium during and after administration of cytochalasin B (0.02 mM). Under these experimental conditions, the intracellular distribution space of D-[(3)H]mannoheptulose averaged 5.42 +/- 0.75 nl/mg in control animals, whereas it failed to be significantly different from zero in the streptozotocin rats. The present procedure may thus allow the assessment of the relative contribution of islet beta-cells to the total mass of the pancreatic gland.

The assessment of antagonist potency under conditions of transient response kinetics.

The muscarinic acetylcholine receptor antagonists, atropine and pirenzepine, produced an apparent insurmountable antagonism of muscarinic M(1) receptor-mediated intracellular Ca(2+) mobilization in Chinese hamster ovary (CHO) cells when tested against the agonists carbachol or xanomeline. Each antagonist caused a dextral shift of the agonist concentration-response curves with depression of the maximum response that was incomplete (i.e., saturated) and which varied with the pairs of agonist and antagonist. Equilibrium competition binding assays found no deviation from simple, reversible competitive behavior for either antagonist. The relative rates of dissociation of unlabeled atropine and pirenzepine were also assessed in radioligand kinetic studies and it was found that atropine dissociated from the receptor approximately 8-fold slower than pirenzepine. Numerical dynamic simulations suggested that the insurmountability of antagonism observed in the present study was probably a kinetic artifact related to the measurement of transient responses to a non-equilibrated agonist in the presence of a slowly dissociating antagonist. Importantly, the patterns of antagonism observed included a saturable depression of agonist maximal response, a mode of antagonism that is incompatible with the previously described phenomenon of hemi-equilibrium states. Monte Carlo simulations indicated that reasonable, semi-quantitative estimates of antagonist potency could be determined by a minor modification of standard methods, where equieffective agonist concentrations, rather than EC(50) values, are compared in the absence and presence of antagonist. Application of the latter approach to the functional data yielded estimates of antagonist potency that were in excellent agreement with those derived from the equilibrium binding assays, thus indicating that the present method can be useful for quantifying antagonist potency under non-equilibrium conditions.

An assessment of the role of intracellular free Ca2+ in E. coli.

We have previously proposed that fluctuations in Ca(2+) levels should play an important role in bacteria as in eukaryotes in regulating cell cycle events (Norris et al., J. Theor. Biol. 134 (1998) 341-350). This proposal implied the presence of Ca(2+) uptake systems in bacteria, cell cycle mutants simultaneously defective in Ca(2+)-homeostasis, and perturbation of cell cycle processes when cellular Ca(2+) levels are depleted. We review the properties of new cell cycle mutants in E. coli and B. subtilis resistant to inhibitors of calmodulin, PKC or Ca(2+)-channels; the evidence for Ca(2+)-binding proteins including Acp and FtsZ; and Ca(2+)-transporters. In addition, the effects of EGTA and verapamil (a Ca(2+) channel inhibitor) on growth, protein synthesis and cell cycle events in E. coli are described. We also describe new measurements of free Ca(2+)-levels, using aequorin, in E. coli. Several new cell cycle mutants were obtained using this approach, affecting either initiation of DNA replication or in particular cell division at non-permissive temperature. Several of the mutants were also hypersensitive to EGTA and or Ca(2+). However, none of the mutants apparently involved direct alteration of a drug target and surprisingly in some cases involved specific tRNAs or a tRNA synthetase. The results also indicate that the expression of several genes in E. coli may be regulated by Ca(2+). Cell division in particular appears very sensitive to the level of cell Ca(2+), with the frequency of division clearly reduced by EGTA and by verapamil. However, whilst the effect of EGTA was clearly correlated with depletion of cellular Ca(2+) including free Ca(2+), this was not the case with verapamil which appears to change membrane fluidity and the consequent activity of membrane proteins. Measurement of free Ca(2+) in living cells indicated levels of 200-300 nM, tightly regulated in wild type cells in exponential phase, somewhat less so in stationary phase, with apparently La(2+)-sensitive PHB-polyphosphate complexes involved in Ca(2+) influx. The evidence reviewed increasingly supports a role for Ca(2+) in cellular processes in bacteria, however, any direct link to the control of cell cycle events remains to be established.

Preoperative assessment of body fluid disturbances in patients with obstructive jaundice.

Postoperative renal dysfunction in obstructive jaundice (OJ) patients has been associated with hypovolemia and depletion of the extracellular water compartment (ECW). The aim of the study was to evaluate the preoperative status of body compartments in OJ patients measured by two methods. In a prospective study 39 OJ patients (11 benign and 28 malignant obstructions) were investigated, with 15 healthy subjects used as a control group (CG). Bioelectrical impedance analysis (BIA) determinations and values derived from anthropometric measurements were used to assess body compartment status. The coefficient of variation of BIA was below 4% in both OJ and CG subjects. No differences were found in intracellular water. However total body water (TBW) and ECW were reduced in OJ patients (50.5 +/- 4.6 vs. 56 +/- 8% body weight, p = 0.05; and 21 +/- 4.5 vs. 23.8 +/- 2.5% body weight, p < 0.05, respectively). There were no differences between benign and malignant obstructions. Seventy four percent of OJ patients had an ECW volume below the mean +/- 2 SD in the CG subjects. Anthropometric and BIA determinations correlated closely for TBW measurements in both CG (r = 0.92, p < 0.001) and OJ patients (r = 0.91, p < 0.001). Bland-Altman analysis also showed that for TBW the BIA was in agreement with anthropometry. In the present study, BIA offered a good correlation with anthropometric determinations and was a reliable method for body fluid disturbances assessment in jaundiced patients.

Cell behaviour as a dynamic attractor in the intracellular signalling system.

We present a model of the cell signalling network based on the generic properties of interactions between protein kinases (PKs) and protein phosphatases (PPs) inside cells. The model is designed to examine the global properties and intrinsic dynamics of the phosphorylation system. A genetic algorithm (GA) is used to evolve populations of "cells". The GA selects cells and ranks them based on an analysis of the dynamics of the proteins within the networks from a series of different random starting conditions. The fittest cells are taken to be those which can generate a variety of different "behaviours" from a series of different initial conditions. During the GA, intracellular protein interactions evolve via mutation and an analogue of domain shuffling between protein types that is thought to occur during biological evolution. The dynamics of the simulated networks are presented and we discuss the hypothesis that changes in the behaviour of a cell may be interpretable as a switch between attractor basins in the intracellular signalling network.

Water signal attenuation in diffusion-weighted 1H NMR experiments during cerebral ischemia: influence of intracellular restrictions, extracellular tortuosity, and exchange.

The "concept of restricted intracellular water diffusion at permeable boundaries", which was recently used to model diffusion-weighted 1H NMR experiments on glioma cells, was applied to measurements on the rat brain in vivo. Combined with the "concept of extracellular tortuosity", various physiological states of the brain were simulated. Hereby, a variable intracellular volume fraction, intracellular exchange time, and extracellular tortuosity factor were considered for young, adult, and ischemic rat brains. The model simulated the cytotoxic shift of extracellular water, changes in membrane permeability and tissue morphology, and was able to explain the diffusion time dependence as well as the non-monoexponentiality of the diffusion attenuation curves. Preliminary diffusion time dependent experiments on the healthy rat brain (1H NMR imaging) agreed well with the theoretical concept. Hereby, the intracellular water signal was separated from extracellular signal contributions by large diffusion weighting. It showed the characteristic of restricted diffusion as well as a signal decay due to the exchange of intracellular water across the plasma membrane. A map of the mean intracellular exchange time for water in living animal brain was determined, and the upper limit in rat brain was evaluated to 15 ms. The presented methods can be applied to correlate local differences in a map of exchange times with tissue morphology and to detect pathological deviations of the exchange time, e.g., during ischemia.

Numerical isotopomer analysis: estimation of metabolic activity.

The use of stable isotopes to analyze intracellular metabolism is a powerful technique because of the wealth of information contained in the distribution of isotopes in key metabolites. We present a new numerical method of using measurements of isotope isomer (isotopomer) distributions to calculate the fluxes through a biochemical reaction network. Nuclear magnetic resonance (NMR) and/or mass spectroscopy can quantify the isotopomers which result from the metabolism of an isotopically enriched substrate. These data can be analyzed via a numerical model of the metabolic network which uses atom-mapping matrices to simplify model construction. The atom-mapping matrices describe the transfer of atoms from reactant to product and the resulting isoopomer balance equations are compact and intuitive. These equations are solved iteratively to determine the unknown intracellular fluxes. Results from the numerical method agreed with an analytical solution developed for the analysis of the tricarboxylic acid cycle in perfused hearts.

An assessment of 31P MRS as a method of measuring pH in rat tumours.

The contribution of extracellular components to the measurement of pHMRS of a variety of rat tumours (nitrosomethyl urea induced mammary tumours, GH3 prolactinomas, Hepatoma 9618a, UA hepatomas and Walker sarcomas) has been assessed. Acid extractable P(i) was between 2.6 and 12.5 mumol/G wet wt depending on tumour type, and of this 53 +/- 4.8% (mean +/- SEM) was MRS-visible. The P(i) content of tumour exudate was 2-3 mM, of interstitial fluid (sampled from a micropore chamber incorporated within a tumour) 1.7 mM, and of blood plasma 1.95 mM. The mean extracellular volumes of the tumours, measured by distribution of 3H2O and [14C]inulin, were 49-55% depending on tumour type and were at least twice that found in normal liver. Calculations suggested that for most tumours with an extracellular volume not exceeding 55%, at least 65% of the P(i)(MRS) signal was derived from intracellular P(i), and thus that pH(MRS) is a measure of pHi. For each tumour type, pHMRS was measured both in 'pulse-acquire' mode at 1.9 T which may include signals from surrounding tissue, and in localized mode at 4.7 T where the signal came uniquely from tumour tissue. The steady state pHMRS was either neutral or on the alkaline side of neutrality (pH range 7.04-7.37). Raised lactate content and decreased buffering capacity (compared to normal tissues) accompanied these neutral to alkaline pH values.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of dopamine and its metabolites in the intracellular and extracellular compartments of the rat striatum after peripheral administration of L-[11C]dopa.

Success in the synthesis of L-3,4-[beta-11C]dihydroxyphenylalanine (L-[11C]DOPA) and its application to positron emission tomography encouraged us to perform radioactive metabolite analyses in rats in an early phase after peripheral injection of L-[11C]DOPA. Following intravenous injection of [11C]DOPA, the radioactivity associated with DOPA and its metabolites was determined in the striatum after decapitation and in striatal extracellular fluid using in vivo brain microdialysis. Without pretreatment, 70-80% of 11C-radioactivity taken up into the striatum was associated with acidic metabolites of dopamine (DA) from 2 to 30 min after administration of L-[11C]DOPA with or without 300 micrograms/kg of unlabelled L-DOPA. In contrast, 80-90% of 11C-radioactivity in the striatum was associated with DOPA and DA after pretreatment with benserazide (25 mg/kg, i.p.) followed by administration of L-[11C]DOPA with or without unlabelled L-DOPA. The radioactivity in the DOPA fraction decreased with time (from 35% of 11C-radioactivity in the striatum at 5 min to 10% at 30 min), but that in the DA fraction increased (from 57% to 68%). The 11C-radioactivity in the extracellular fluid determined by brain microdialysis was less than 0.4% of that in the whole striatum and no radioactivity was present in the DA fraction. These results suggest that, in an early phase after administration of L-[11C]DOPA, [11C]DA is the main metabolite and is localized exclusively in the intracellular compartment within this time frame.