Cell Energy Budget Platform for Multiparametric Assessment of Cell and Tissue Metabolism.

Specific bioenergetic signature reports on the current metabolic state of the cell, which may be affected by metabolic rearrangement, dysfunction or dysregulation of relevant signaling pathways, altered physiological condition or energy stress. A combined analysis of respiration , glycolytic flux, Krebs cycle activity, ATP levels, and total biomass allows informative initial assessment. Such simple, high-throughput, multiparametric methodology, called cell energy budget (CEB ) platform, is presented here and demonstrated with particular cell and tissue models. The CEB uses a commercial fluorescent lanthanide probe pH-Xtra™ to measure extracellular acidification (ECA) associated with lactate (L-ECA) and combined lactate/CO2 (T-ECA), a phosphorescent probe MitoXpress®-Xtra to measure oxygen consumption rate (OCR), a bioluminescent ATP kit, and an absorbance-based total protein assay. All the assays are performed on a standard multi-label reader. Using the same readouts, the CEB approach can be extended to more detailed mechanistic studies, by targeting specific pathways in cell bioenergetics and measuring other cellular parameters, such as NAD(P)H, Ca2+, mitochondrial pH, membrane potential, redox state, with conventional fluorescent or luminescent probes.

Numerical Modeling of Electroosmotic Push-Pull Perfusion and Assessment of Its Application to Quantitative Determination of Enzymatic Activity in the Extracellular Space of Mammalian Tissue.

Many sampling methods have been developed to measure the extracellular concentrations of solutes in the extracellular space of mammalian tissue, e.g., brain. However, few have been used to quantitatively study the various processes, such as enzymatic degradation, that determines the fate of these solutes. For a method to be useful in this pursuit, it must be able to (1) perfuse tissue and collect the perfusate for quantitative analysis of the solutes introduced and reaction products produced, (2) control the average residence time of the active solutes, and (3) have the appropriate spatial resolution for the process of interest. Our lab previously developed a perfusion technique based on electroosmosis (EO), called EO push-pull perfusion (EOPPP), that is in principle suitable to meet these needs. However, much like the case for other sampling methods that came before, there are parameters that are needed for quantitative interpretation of data but that cannot be measured easily (or at all). In this paper, we present a robust finite element model that provides a deep understanding of fluid dynamics and mass transport in the EOPPP method, assesses the general applicability of EOPPP to studying enzyme activity in the ECS, and grants a simple approach to data treatment and interpretation to obtain, for example, Vmax and Km for an enzymatic reaction in the extracellular space of the tissue. This model is a valuable tool in optimizing and planning experiments without the need for costly experiments.

Effect of citric acid induced deflocculation on the ultrasonic pretreatment efficiency of dairy waste activated sludge.

In this investigation, the application of citric acid was explored for the removal of extracellular polymeric substance (EPS) from waste activated sludge (WAS), followed by ultrasonic pretreatment, which enhanced the subsequent anaerobic biodegradability. EPS was removed with 0.05g/g SS of citric acid. The chemical oxygen demand (COD) solubilization and suspended solids (SS) reduction that occurred for specific energy input of 171.9kJ/kg TS, in deflocculated (EPS removed and ultrasonically pretreated) sludges were found to be 22.70% and 20.28% and was comparatively higher, than the flocculated (with EPS and ultrasonically pretreated). The biogas yield potential of flocculated and deflocculated sludges (specific energy input - 171.9kJ/kgTS) was found to be 0.212L/(gVS) and 0.435L/(gVS), respectively. Accordingly, the deflocculation and ultrasonic pretreatment improved the anaerobic biodegradability efficiently. Thus, this chemo mediated sonic pretreatment is an effective method for enhancing biodegradability and improving clean energy generation from WAS.

Bioelectrical impedance analysis for assessment of fluid status and body composition in neonates--the good, the bad and the unknown.

BACKGROUND/OBJECTIVES: There is a critical need for improved technologies to monitor fluid balance and body composition in neonates, particularly those receiving intensive care. Bioelectrical impedance analysis meets many of the criteria required in this environment and appears to be effective for monitoring physiological trends. SUBJECT/METHODS: The literature regarding the use of bioelectrical impedance in neonates was reviewed. RESULTS: It was found that prediction equations for total body water, extracellular water and fat-free mass have been developed, but many require further testing and validation in larger cohorts. Alternative approaches based on Hanai mixture theory or vector analysis are in the early stages of investigation in neonates. CONCLUSIONS: Further research is required into electrode positioning, bioimpedance spectroscopy and Cole analysis in order to realise the full potential of this technology.

Validity of extracellular water assessment with saliva samples using plasma as the reference biological fluid.

Extracellular water (ECW) assessment is based on dilution techniques, commonly using blood sampling. However, plasma collection is an invasive procedure. We aimed to validate the use of saliva for ECW estimation by the bromide dilution technique using plasma as the reference method, in a sample of elite athletes. A total of 89 elite athletes with a mean age of 20.4 ± 4.4 years were evaluated. Baseline samples were collected before sodium bromide oral dose administration, and enriched samples were collected 3 h post-dose administration. The bromide concentration was assessed by high-performance liquid chromatography. Comparison of means, concordance coefficient correlation (CCC), multiple regression and Bland-Altman analysis were performed. The ECW from saliva explained 91% of the variance in ECW by plasma with a standard error of estimation of 0.91 kg. The CCC between alternative and reference methods was 0.952. No significant trend was observed between the mean and difference of the methods, with limits of agreement ranging between -1.5 and 2.1 kg. These findings reveal that bromide dilution volume calculated from saliva samples is a valid noninvasive method for ECW assessment in elite athletes.

Ion trapping with fast-response ion-selective microelectrodes enhances detection of extracellular ion channel gradients.

Previously, functional mapping of channels has been achieved by measuring the passage of net charge and of specific ions with electrophysiological and intracellular fluorescence imaging techniques. However, functional mapping of ion channels using extracellular ion-selective microelectrodes has distinct advantages over the former methods. We have developed this method through measurement of extracellular K+ gradients caused by efflux through Ca2+-activated K+ channels expressed in Chinese hamster ovary cells. We report that electrodes constructed with short columns of a mechanically stable K+-selective liquid membrane respond quickly and measure changes in local [K+] consistent with a diffusion model. When used in close proximity to the plasma membrane (<4 microm), the ISMs pose a barrier to simple diffusion, creating an ion trap. The ion trap amplifies the local change in [K+] without dramatically changing the rise or fall time of the [K+] profile. Measurement of extracellular K+ gradients from activated rSlo channels shows that rapid events, 10-55 ms, can be characterized. This method provides a noninvasive means for functional mapping of channel location and density as well as for characterizing the properties of ion channels in the plasma membrane.

[Extra cellular volume assessment methods in dialysis, a critical analysis]. / Analyse critique des méthodes de mesures du volume extra-cellulaire en dialyse.

The extra cellular volume is strictly proportional to the amount of sodium present in the body. Its clinical evaluation on dialysis (through clinical story, weight and blood pressure changes) often needs probing for dry weight by reducing progressively the post dialysis weight down to the point where hypotension regularly occurs. Common critics addressed to the clinical assessment of dry weight include lack of sensitivity, of objectivity and of repeatability. But non clinical methods are far from perfect. They are non invasive, but they are poorly sensitive and reproducible (except for bio impedance, but in very strictly equal operational conditions). Most of them (natriuretic peptides, inferior vena cava echography, on-line volemia) measure exclusively the plasma volume but not the extra cellular volume. Besides, they increase dialysis complexity and cost. None of them is so far validated for dialysis. The accurate and absolute value of extra cellular volume is useless in clinical daily practice. What is needed is a simple fast evaluation of the actual extra cellular volume relative to its ideal level (dry weight). The clinical method based on two very simple, costless objective measurements, weight and blood pressure, allows for fulfilling the goal, the continuous adjustment of extra cellular volume and blood pressure normalization.

In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life.

Soluble amyloid-beta (Abeta) peptide converts to structures with high beta-sheet content in Alzheimer's disease (AD). Soluble Abeta is released by neurons into the brain interstitial fluid (ISF), in which it can convert into toxic aggregates. Because assessment of ISF Abeta levels may provide unique insights into Abeta metabolism and AD, an in vivo microdialysis technique was developed to measure it. Our Abeta microdialysis technique was validated ex vivo with human CSF and then in vivo in awake, freely moving mice. Using human amyloid precursor protein (APP) transgenic mice, we found that, before the onset of AD-like pathology, ISF Abeta in hippocampus and cortex correlated with levels of APP in those tissues. After the onset of Abeta deposition, significant changes in the ISF Abeta40/Abeta42 ratio developed without changes in Abeta1-x. These changes differed from changes seen in tissue lysates from the same animals. By rapidly inhibiting Abeta production, we found that ISF Abeta half-life was short ( approximately 2 hr) in young mice but was twofold longer in mice with Abeta deposits. This increase in half-life, without an increase in steady-state levels, suggests that inhibition of Abeta synthesis reveals a portion of the insoluble Abeta pool that is in dynamic equilibrium with ISF Abeta. This now measurable in vivo pool is a likely target for new diagnostic and therapeutic strategies.

Assessment of the role of interstitial glucagon in the acute glucose secretory responsiveness of in situ pancreatic beta-cells.

Glucagon is a potent stimulator of insulin release in the presence of a permissive glucose concentration, activating beta-cells in vitro via both glucagon- and glucagon-like peptide-1 (GLP-1)-receptors. It is still unclear whether locally released glucagon amplifies the secretory responsiveness of neighboring beta-cells in the intact pancreas. The present study investigates this question in the perfused pancreas by examining the effects of antagonists for glucagon receptors ([des-His(1),des-Phe(6),Glu(9)]glucagon-NH(2), 10 micromol/l) and GLP-1-receptors [exendin-(9-39)-NH(2), 1 micromol/l] on the insulin secretory response to glucose. The specificity of both antagonists was demonstrated by their selective interaction with glucagon-receptor signaling in rat hepatocytes and GLP-1-receptor signaling in Chinese hamster lung (CHL) fibroblasts. In purified rat beta-cells, the glucagon-receptor antagonist (10 micromol/l) inhibited the effect of 1 nmol/l glucagon upon glucose-induced insulin release by 78 plus minus 6%. In the perfused rat pancreas, neither of these antagonists inhibited the potent secretory response to 20 mmol/l glucose, although they effectively suppressed the potentiating effect of, respectively, an infusion of glucagon (1 nmol/l) or GLP-1 (1 nmol/l) on insulin release. When endogenous glucagon release was enhanced by isoproterenol (100 nmol/l), no amplification was seen in the simultaneous or subsequent insulin secretory response to glucose. It is concluded that, at least under the present selected conditions, the glucose-induced insulin release by the perfused rat pancreas seems to occur independent of an amplifying glucagon signal from neighboring alpha-cells.

In vivo assessment of free radical activity during shock wave lithotripsy using a microdialysis system: the renoprotective action of allopurinol.

PURPOSE: Shock wave lithotripsy is believed to cause renal damage directly through cellular injury from high energy shock waves and indirectly through vascular injury and resultant ischemia, which gives rise to oxygen free radical compounds. The transient and volatile nature of free radicals and derived products makes their detection difficult. Moreover, certain medications may provide a protective effect against shock wave lithotripsy induced renal parenchymal injury. We introduced an innovative microdialysis system for in vivo sampling of interstitial fluids that can be analyzed for free radical mediated lipid peroxidation products after shock wave lithotripsy treatment in the swine model. In addition, this system was used to test the antioxidant or renoprotective action of allopurinol. MATERIALS AND METHODS: Ten juvenile swine were assigned to a nonmedicated control group that underwent shock wave lithotripsy or to a group that was premedicated with allopurinol before shock wave lithotripsy. Each group of animals underwent shock wave lithotripsy to the lower pole of the right kidney and received a total of 10,000 shock waves. Dialysate fluid was collected at 1,000-shock wave increments via probes surgically implanted into the lower pole of the right and left kidneys before lithotripsy. Samples were immediately preserved in liquid nitrogen and subsequently analyzed for the presence and concentration of conjugated diene levels, a measure of lipid peroxidation. Five additional juvenile swine were assigned to a sham treated group that did not undergo shock wave lithotripsy. Dialysate fluid was collected from the lower pole of the right and left kidneys to establish baseline or pre-lithotripsy levels of conjugated dienes. RESULTS: After shock wave lithotripsy conjugated diene levels increased almost 100-fold over that in the right kidneys of the nonmedicated control group. The difference was statistically significant compared to levels in the contralateral untreated kidneys (p <0.01). Right kidneys in the group premedicated with allopurinol did not demonstrate an increase in conjugated diene levels during shock wave lithotripsy. CONCLUSIONS: The results of this study confirm shock wave lithotripsy induced free radical activity as well the antioxidant and protective nature of allopurinol. The newly described microdialysis system enables real-time sampling of interstitial fluids during shock wave lithotripsy. It represents a unique method for assessing free radical formation and evaluating the protective effects of additional antioxidant medications.

Effects of short-term carbohydrate or fat overfeeding on energy expenditure and plasma leptin concentrations in healthy female subjects.

OBJECTIVE: To determine the effects of excess carbohydrate or fat intake on plasma leptin concentrations and energy expenditure. DESIGN: Ten healthy lean females were studied: (a) during a 3 day isoenergetic diet (ISO); (b) during 3 day carbohydrate overfeeding (CHO OF); and (c) during 3 day fat overfeeding (FAT OF). During each test, basal metabolic rate, the energy expended during mild physical activity and recovery, and 24 h energy expenditure (24 h EE) were measured with indirect calorimetry. The concentrations of glucose and lactate were monitored in subcutaneous interstitial fluid over a 24 h period using microdialysis. Plasma hormone and substrate concentrations were measured in a blood sample collected in the morning of the fourth day. RESULTS: CHO OF increased plasma leptin concentrations by 28%, and 24 h EE by 7%. Basal metabolic rate and the energy expended during physical activity were not affected. FAT OF did not significantly change plasma leptin concentrations or energy expenditure. There was no relationship between changes in leptin concentrations and changes in energy expenditure, suggesting that leptin is not involved in the stimulation of energy metabolism during overfeeding. Interstitial subcutaneous glucose and lactate concentrations were not altered by CHO OF and FAT OF. CONCLUSIONS: CHO OF, but not FAT OF, increases energy expenditure and leptin concentration.

Can interstitial glucose assessment replace blood glucose measurements?

Current treatment regiments for individuals depending on exogenous insulin are based on measurements of blood glucose obtained through painful finger sticks. The shift to minimal or noninvasive continuous glucose monitoring primarily involves a shift from blood glucose measurements to devices measuring subcutaneous interstitial fluid (ISF) glucose. As the development of these devices progresses, details of the dynamic relationship between blood glucose and interstitial glucose dynamics need to be firmly established. This is a challenging task insofar as direct measures of ISF glucose are not readily available. The current article investigated the dynamic relationship between plasma and ISF glucose using a model-based approach. A two-compartment model system previously validated on data obtained with the MiniMed Continuous Glucose Monitoring System (CGMS) is reviewed and predictions from the original two-compartment model were confirmed using new data analysis of glucose dynamics in plasma and hindlimb lymph (lymph is derived from ISF) in the anesthetized dog. From these data sets, the time delay between plasma and ISF glucose in dogs was established (5-12 minutes) and a simulation study was performed to estimate the errors introduced if ISF is taken as a surrogate for blood. From the simulation study, the error component resulting from the differences in plasma and ISF glucose was estimated to be < 6% during normal day-to-day use in an individual with diabetes (error component calculated as the standard deviation of the ISF/plasma glucose differences under conditions where the maximal time delay was used). This difference is most likely within the variance between arterial and venous blood glucose. We conclude that the differences between plasma and ISF glucose will not be a significant obstacle in advancing the use of ISF as an alternative to blood glucose measurements.

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.

Assessment of the protein quality of the smooth muscle myofibrillar and connective tissue proteins of chicken gizzard.

The objective of this study was to assess the protein quality of the myofibrillar and connective tissue proteins of chicken gizzard. Protein fractions were isolated from White Leghorn chicken gizzards and quantified by detailed amino acid analysis. This quantification involved repeated extractions of ground gizzards first with Triton X-100, then with low ionic strength imidazole-buffered saline (pH 7.1), followed by either 2% SDS or by 5 M guanidine hydrochloride. The total soluble intracellular protein fraction averaged 86.3% of the total protein and the insoluble extracellular connective tissue proteins comprised the remaining 13.7%. These fractions differed significantly in their essential amino acid (EAA) profiles, with the soluble intracellular fraction having the highest percentage EAA9 (48.6 to 49.0%) and the insoluble connective tissue fraction varying from 20.8 to 23%, compared to the FAO/WHO reference pattern value of 33.9% for a 2- to 5-yr-old child. Calculated protein efficiency ratios (PER) for intracellular proteins averaged 3.02 compared with a value of 1.65 for the extracellular matrix proteins. These results provide an accurate assessment of the protein quality of smooth muscle proteins of chicken gizzard and may prove valuable for industrial control of the amount of connective tissue added to formulations of meats and poultry products.

The importance of the measurement of ATP depletion and subsequent cell damage with an estimate of size and nature of the market for a practicable method: a review designed for technology transfer.

ATP is the energy currency of cells. ATP depletion is a central process in pathogenesis, in particular ischaemia, hypoxia and hypoglycaemia. ATP depletion in cells can be indirectly measured from the increased concentrations of extracellular hypoxanthine, a central intermediate in the metabolism of ATP. Cell damage secondary to ATP depletion can also be measured from extracellular hypoxanthine. The relevant biochemistry and physiology is briefly reviewed. Since market size is needed for investment decisions that would allow technology transfer, the numbers of hypoxanthine analyses that are clinically justified from the extensive published evidence are calculated per million population from UK, Norwegian and other evidence. The concentration of oxygen in blood is measured to estimate whether mitochondrial oxidative phosphorylation is adequate. Measurements of bicarbonate are used to estimate anaerobic glycolysis. Since the indirect estimation of ATP depletion is a major objective of blood gas and acid-base analyses, the number of such analyses per million population provides a good estimate of potential market size for a more direct method of estimating ATP depletion. A method is required for the rapid, dispersed emergency analyses needed clinically. Routes for method development are indicated. Competition, risks, acceptability, consumer motivation and timetables are indicated for the development phase. There are medicolegal pressures, especially in the USA, for the proposed advances to be widely used.

In vivo monitoring of brain neurotransmitter release for the assessment of neuroendocrine interactions.

1. The neurotransmitter mechanisms regulating neuroendocrine processes have been traditionally inferred from the effects of drugs purportedly acting through specific transmitter systems. The direct appraisal of changes in endogenous neuromediators had to rely initially on analyses of brain samples obtained post-morten. 2. Currently, a more physiological assessment is available through the monitoring ot the extracellular levels of neurotransmitters and their metabolites in discrete brain areas of living animals. Two methodologies, namely in vivo voltammetry and microdialysis, are being increasingly used for this purpose. This article summarizes their principles, relative merits, and limitations and presents some relevant applications. 3. Thus, microdialysis data show a differential response in the amphetamine-induced dopamine release in the nucleus accumbens in adult male and female rats castrated prepuberally. Given their high time-resolution, in vivo electrochemistry techniques seem especially suited for studying the fast, non-genomic effects of steroid hormones. This is illustrated by the voltammetric detection of a rapid release of dopamine in the corpus striatum induced by progesterone in males. 4. These methodologies should be regarded as complementary tools for the assessment of the neurochemical correlates of neuroendocrine interactions.

Comparison and assessment of blood gas related quantities including base excess, the gas exchange indices, and temperature corrected pH/PO2/PCO2, as defined in approved NCCLS standard C12-A, using a computer simulation of input variables.

Blood gases and related quantities reported to clinicians have, since the earliest days, included both directly measured as well as calculated or estimated quantities. Some developed as substitutes for quantities that were or are difficult to measure routinely, others to explain relationships between older, difficult to measure quantities and newly measureable quantities, and still others attempt to better understand the physiology of the acid-base process. The net result is a plethora of acid-base and related quantities that may be reported by different blood gas systems. In an attempt to address the issue of which quantities have stood the test of usefulness over time, and further, to examine the optimum algorithm for use in quantification, the NCCLS has developed, through its consensus process, a recommended set of quantities and their quantifying algorithms. We have studied these quantities and compared them with some other recognized approaches and present our analysis in this report. The major conclusion is that among those quantities recommended, the NCCLS algorithms present the most sensible overall approach and that we would recommend their use as described so that the quantities can be most effectively applied clinically, without differences in final values occurring due solely simple algorithm differences.

In vivo total body water assessment by total body electrical conductivity in rats suffering perturbations of water compartment equilibrium.

Total body electrical conductivity (TOBEC) is a simple and non-invasive method for the assessment of body composition in vivo. Information regarding the applicability of TOBEC in the condition of abnormal fluid balance is scarce. In the present paper we give the results of the comparison between TOBEC and total body water (TBW; assessed by the tritium dilution technique) in three groups of animals: (1) healthy (n 17), (2) expanded fluid volume by secondary biliary cirrhosis (SBC; n 9) and (3) Furosemide-treated rats (n 9). The TOBEC score and TBW by tritium dilution were found to be highly correlated in the pooled sample (r 0.90) and in normal (r 0.87), SBC (r 0.73) and Furosemide-treated (r 0.89) rats. However, the relationship between TOBEC and TBW, described by least-squares regression analysis, was found to be similar for SBC and normal rats but was significantly different for Furosemide-treated and normal rats. These findings suggest that TOBEC is unable to track TBW accurately when the ratio between intracellular and extracellular water is chronically or acutely altered.