High temporal resolution delayed analysis of clinical microdialysate streams.

This paper presents the use of tubing to store clinical microdialysis samples for delayed analysis with high temporal resolution, offering an alternative to traditional discrete offline microdialysis sampling. Samples stored in this way were found to be stable for up to 72 days at -80 °C. Examples of how this methodology can be applied to glucose and lactate measurement in a wide range of in vivo monitoring experiments are presented. This paper presents a general model, which allows for an informed choice of tubing parameters for a given storage time and flow rate avoiding high back pressure, which would otherwise cause the microdialysis probe to leak, while maximising temporal resolution.

Assessment of tissue viability following electroosmotic push-pull perfusion from organotypic hippocampal slice cultures.

We have developed a novel sampling technique that allows both introduction and removal of fluid from the extracellular space of living tissue. This method is based on the fluidics of push-pull perfusion but flow is driven by electroosmosis. We have applied this method to organotypic hippocampal cultures. A source capillary is inserted into the tissue and a collection capillary is in contact with the tissue surface through a thin layer of fluid. A voltage is applied across the proximal ends of source and collection capillary. In the applied field, fluid will move from source, into the tissue, and then be collected. In this process, damage to cells may occur. To understand better what sampling conditions influence damage most, we tested various sampling geometries and applied voltages, quantifying damage 16-24 h later using propidium iodide as a cell death marker. We found that damage correlates with both voltage drop and power dissipated in the tissue, but that voltage drop is a better indicator of damage when comparing models in which capillary arrangement and length are different.

Electroosmotic push-pull perfusion: description and application to qualitative analysis of the hydrolysis of exogenous galanin in organotypic hippocampal slice cultures.

We demonstrate here a method that perfuses a small region of an organotypic hippocampal culture with a solution containing an enzyme substrate, a neuropeptide. Perfusate containing hydrolysis products is continually collected and subsequently analyzed for the products of the enzymatic degradation of the peptide substrate. The driving force for perfusion is an electric field. The fused silica capillaries used as "push" and "pull" or "source" and "collection" capillaries have a ζ-potential that is negative and greater in magnitude than the tissue's ζ-potential. Thus, depending on the magnitudes of particular dimensions, the electroosmotic flow in the capillaries augments the fluid velocity in the tissue. The flow rate is not directly measured; however, we determine it using a finite-element approach. We have determined the collection efficiency of the system using an all d-amino acid internal standard. The flow rates are low, in the nL/min range, and adjustable by controlling the current or voltage in the system. The collection efficiency of the d-amino acid peptide internal standard is variable, increasing with increased current and thus electroosmotic flow rate. The collection efficiency can be rationalized in the context of a Peclet number. Electroosmotic push-pull perfusion of the neuropeptide galanin (gal1-29) through the extracellular space of an organotypic hippocampal culture results in its hydrolysis by ectopeptidase reactions occurring in the extracellular space. The products of hydrolysis were identified by MALDI-MS. Experiments at two levels of current (8-12 µA and 19-40 µA) show that the probability of seeing hydrolysis products (apparently from aminopeptidases) is greater in the Cornu Ammonis area 3 (CA3) than in the Cornu Ammonis area 1 (CA1) in the higher current experiments. In the lower current experiments, shorter peptide products of aminopeptidases (gal13-29 to gal20-19) are seen with greater frequency in CA3 than in CA1 but there is no statistically significant difference for longer peptides (gal3-29 to gal12-29).

Electroporation of single cells and tissues with an electrolyte-filled capillary.

We show how an electrolyte-filled capillary (EFC) coupled to a high-voltage power supply can be used as a versatile electroporation tool for the delivery of dyes, drugs, and biomolecules to the cytoplasm of single cells and cells in tissues. A large-voltage pulse applied across the EFC (fused silica, 30 cm long, 375-microm o.d., 30-microm i.d.) gives rise to a small electric field outside the terminus of the EFC, which causes pore formation in cell membranes and induces an electroosmotic flow of electrolyte. When the EFC contains cell-loading agents, then the electroosmotic flow delivers the agents at the site of pore formation. The combination of pore formation and delivery enables loading of materials into the cytoplasm. By patch-clamp and fluorescence microscopy, formation of pores was observed at estimated transmembrane voltages of <85 mV with half-maximum values around 206 mV. The electroporation protocol was demonstrated by introduction of fluorogenic dyes into single NG108-15 cells, cellular processes, and small populations of cells in organotypic hippocampal cultures. Preliminary results are shown in which this protocol was employed for in vivo electroporation of ventral mesencephalon in rat brains. The technique was also used to access organelle-based detection systems inside cells. As a demonstration, 1,4,5-inositoltriphosphate was added to the electrolyte and detected by intracellular organelles in electroporated cells.

Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy.

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized.

Chromatographic detection of nitroaromatic and nitramine compounds by electrochemical reduction combined with photoluminescence following electron transfer.

The oxidizing agent tris(bipyridyl)ruthenium(III), or Ru-(bpy)(3)3+, is used as a postcolumn reagent for the detection of nitroaromatic and nitramine explosive compounds. After separation, the explosives are reduced electrochemically to oxidizable products such as hydroxlamines and nitrosamines, and these products react readily with Ru-(bpy)(3)3+ and Ru(bpy)(3)2+. The photoluminescence from the latter is used for detection. A porous carbon electrode was used for on-line analyte reduction following chromatography. Another porous carbon electrode was used to generate the nonluminescent Ru(bpy)(3)3+ from Ru(bpy)(3)3+ on-line at high efficiency. The two streams were combined, and the Ru(bpy)(3)2+ produced by oxidation of the reduced analytes was detected by laser illumination and light detection. Reductive hydrodynamic voltammograms of nitrobenzene, 2,4,6-trinitrotoluene, and hexahydro-1,3,5-trinitro-1,3,5-triazine indicated that a potential of - 1500 mV vs Ag/AgCl was sufficient to achieve a maximum signal from the reduced analytes. HPLC with a water/acetonitrile gradient on a C-18 reversed-phase column was then used to determine these three compounds plus the four additional examples, 1,3,5,7-tetrazocine, 2,4-dinitrotoluene; 2,6-dinitrotoluene, and 4-nitrotoluene. For both hydrodynamic voltammetry and HPLC detection, the photoluminescence following electron-transfer signal was calibrated using the one-electron standards ferrocene and ferrocenecarboxylic acid. Detection limits were in the low-nanomolar range for 20-microL injections of nonpreconcentrated nitro compounds.

Alterations in glutathione and amino acid concentrations after hypoxia-ischemia in the immature rat brain.

Hypoxic-ischemic brain injury involves an increased formation of reactive oxygen species. Key factors in the cellular protection against such agents are the GSH-associated reactions. In the present study we examined alterations in total glutathione and GSSG concentrations in mitochondria-enriched fractions and tissue homogenates from the cerebral cortex of 7-day-old rats at 0, 1, 3, 8, 14, 24 and 72 h after hypoxia-ischemia. The concentration of total glutathione was transiently decreased immediately after hypoxia-ischemia in the mitochondrial fraction, but not in the tissue, recovered, and then decreased both in mitochondrial fraction and homogenate after 14 h, reaching a minimum at 24 h after hypoxia-ischemia. The level of GSSG was approximately 4% of total glutathione and increased selectively in the mitochondrial fraction immediately after hypoxia-ischemia. The decrease in glutathione may be important in the development of cell death via impaired free radical inactivation and/or redox related changes. The effects of hypoxia-ischemia on the concentrations of selected amino acids varied. The levels of phosphoethanolamine, an amine previously reported to be released in ischemia, mirrored the changes in glutathione. GABA concentrations initially increased (0-3 h) followed by a decrease at 72 h. Glutamine levels increased, whereas glutamate and aspartate were unchanged up to 24 h after the insult. The results on total glutathione and GSSG are discussed in relation to changes in mitochondrial respiration and microtubule associated protein-2 (MAP2) which are reported on in accompanying paper [64].

Glutathione efflux induced by NMDA and kainate: implications in neurotoxicity?

Neurotoxicity in acute as well as chronic neurological diseases may be partly mediated by oxidative stress caused by overactivation of glutamate receptors. A key component of the cellular defense against oxidative stress is reduced glutathione. In our earlier work, we have shown that ischemia in brain induces increased efflux, elevated metabolism, and decreased tissue concentrations of glutathione. In this study, we have evaluated the effect of glutamate receptor activation on the efflux of glutathione from hippocampus in vitro. NMDA and kainate induced a delayed increase in glutathione, taurine, and phosphoethanolamine efflux. Extracellular glutathione was recovered mainly in the reduced form (85-95%); the efflux was dependent on extracellular calcium but unrelated to dantrolene-sensitive intracellular calcium release and independent of glutathione or NO synthesis. The NMDA-induced efflux of glutathione was enhanced by blockage of gamma-glutamyl transpeptidase, indicating an increased transpeptidation of glutathione after NMDA receptor activation. Our results suggest that increased efflux of glutathione could be a factor in initiating nerve cell death via a change in intracellular redox potential and/or a decrease in the intracellular capacity for inactivation of reactive oxygen species.

Chromatographic detection using tris(2,2'-bipyridyl)ruthenium(III) as a fluorogenic electron-transfer reagent.

Tris(2,2'-bipyridyl)ruthenium can be excited to fluorescence by visible light (lambda abs 454 nm, lambda em 607 nm) when in the M(II) oxidation state, but not in the M(III) state. A novel chromatographic detection method using the non-fluorescent M(III) form of the complex as a postcolumn fluorogenic reagent is demonstrated. The M(III) form is a powerful oxidizing agent (E degree = 1.27 V vs NHE, 1.05 V vs Ag/AgCl). The M(III) reagent is generated on-line from the M(II) form of the complex by a highly efficient porous carbon electrode and then reacted briefly with chromatographic effluent; the M(II) created by electron transfer from oxidation-susceptible analytes is then detected by fluorescence. The fluorescence detector can be calibrated for number of electrons transferred by injection of either M(II) or an oxidative standard such as ferrocyanide. It is hoped that this redox-based detection scheme will provide an alternative to electrochemical detection. Among the advantages are freedom from surface fouling and the potential for extremely low detection limits. The scheme was applied to detection of the peptide dynorphin A and several of its fragments. Dynorphin A contains tyrosine at the N-terminus (position 1) and tryptophan in position 15; these amino acid residues are susceptible to oxidation and peptides containing them can be detected on that basis. Flow injection testing of the model compounds Tyr-Gly-Gly-Phe-Leu and Gly-Gly-Trp-Gly indicated that tyrosine transferred approximately 1 electron to the M(III) reagent and that tryptophan transferred approximately 4 electrons. Similar results were obtained from the chromatographic runs. Dynorphin A and six dynorphin A fragments containing the N-terminal tyrosine were detected easily at 100 nM concentration (14 pmol) using laser-induced fluorescence. As expected, one fragment that did not contain tryptophan or tyrosine was not detected. A mass detection limit of 80 fmol was estimated for the tyrosine-containing fragments.

Net efflux of cysteine, glutathione and related metabolites from rat hippocampal slices during oxygen/glucose deprivation: dependence on gamma-glutamyl transpeptidase.

Extracellular metabolism of the protective substance glutathione (gamma-glutamyl-cysteinyl-glycine) may generate cysteine, glycine, several gamma-glutamyl-containing dipeptides and possibly free glutamate, all of which could participate in neurotoxicity. In the present study, we have examined how blockage of gamma-glutamyl transpeptidase, the key enzyme in glutathione degradation, influences the extracellular concentrations of glutathione, cysteine and related metabolites during anoxia/aglycemia of rat hippocampal slices. The net efflux, i.e., the increase in extracellular concentration due to changes in release and/or uptake, of cysteine, cysteine sulfinate, gamma-glutamyl-glutamate, gamma-glutamyl-glutamine, glutathione, gamma-glutamyl-cysteine and glutamate increased as a result of anoxia/aglycemia. These increases in net efflux of cysteine, cysteine sulfinate, gamma-glutamyl-glutamate and gamma-glutamyl-glutamine were reduced or blocked by acivicin, an inhibitor of gamma-glutamyl transpeptidase. In contrast, acivicin caused an increase in both basal and anoxia/aglycemia-induced net efflux of glutathione whereas the basal and anoxia/aglycemia-induced efflux of glutamate was unchanged by acivicin treatment. The effect of acivicin on the efflux of gamma-glutamyl-cysteine was similar to that of glutathione although less pronounced. Addition of beta-mercaptoethanol to the incubation medium during and after 30 min of anoxia/aglycemia decreased the net efflux of cysteine sulfinate specifically, indicating that the increase in cysteine sulfinate during anoxia/aglycemia may be partly derived from the spontaneous oxidation of cysteine. The results suggest that gamma-glutamyl transpeptidase may be involved in the regulation of the extracellular concentrations of cysteine, several gamma-glutamyl-containing dipeptides and glutathione but not glutamate during ischemia.

Development of a liquid chromatographic method for picomole determination of S-sulfocysteine in trifluoroacetic acid extracts of neonatal rat brain.

Neonatal Sprague Dawley rat brain tissue was extracted with methanol, acetonitrile, acetic acid and trifluoroacetic acids (TFA). Among the extractants tested, 0.1 M TFA gave the highest recovery, 73.4 +/- 5.2% (slope of regression of 'added' vs. 'found' and standard error of the slope) of S-sulfocysteine (SSC). The poorest recovery of SSC was found with acetonitrile and 90% methanol extractions (less than 10%). Possible reasons for the low recoveries have been explored. The recovery of SSC from aqueous standards in 0.1 M TFA is 92 +/- 5%. Detection of picomole quantities of SSC has been demonstrated with a combination of the optimized extraction procedures and our previously developed detection system. Supernatant of rat brain homogenate (0.10 M TFA as extractant) was evaporated to dryness in a vacuum centrifuge. Residues were reconstituted with deionized water. Samples were separated on a reversed phase column. The mobile phase was 20 mM aqueous acetate buffer (pH 5.2) containing 0.40 mM cetyl trimethylammonium p-toluene sulfonate and 2 vol.% methanol. Electrochemical detection used dual series gold-mercury amalgam electrodes. For the first time, S-sulfocysteine was detected in normal neonatal rat brain. Its concentration is 0.99 +/- 0.25 pmol/mg brain tissue. The results indicate that TFA, rarely reported an an extractant, efficiently recovers SSC from rat brain tissues.

Artificial receptor-facilitated solid-phase microextraction of barbiturates.

A receptor for barbiturates, N,N'-Bis-[6-(2-ethylhexanoylamino)-pyridin-2-yl]-isophthalamide, was designed to dissolve in plasticizers of poly(vinyl chloride) (PVC). Microextractions using receptor-doped films of PVC were carried out as a function of receptor concentration. The effect of the concentration of the receptor on extraction yield is considerable for barbiturates that have significant binding to the receptor but negligible for very similar molecules that do not bind to the receptor strongly. Thus, it is the receptor's ability in molecular recognition, not its generic ability as an H-bonding cosolvent, that is important. On the other hand, NMR data show that the receptor self-associates. A simple, approximate analysis is given to extract the amount of active receptor from the data. Receptor-enhanced extractions of barbiturates from urine are compared to extractions using a phosphate ester as solvent.

Comparison of anion-exchange and ion-modified reversed-phase liquid chromatography for the determination of S-sulfocysteine.

A dual Hg-Au amalgam electrode is used to detect S-sulfocysteine (SSC) in this study. There exist two main components in the acetonitrile (ACN) rat brain extracts, namely, Cl- and GSSG (oxidized glutathione), that are active in our detection system (GSH is not extracted in ACN). Two strong anion-exchange columns from different companies were used to separate the samples under different conditions, but SSC and Cl- were not separated at the optimum detection pH of 5.2. The signal from Cl- was greatly decreased by lowering the potential at the downstream electrode, though it cannot be completely eliminated. While a silver cartridge removed Cl- from micromoles to several millimoles without any negative effect on the SSC signal in aqueous standards, a large negative peak which interferes with SSC detection was unfortunately introduced when a silver cartridge was applied to brain tissue samples. However, SSC and Cl- in the samples are successfully separated by ion-modified reversed-phase LC in acetate buffer at the optimum detection pH (5.2). The separation conditions are 20 mM acetic acid, 2% methanol, 0.5 mM cetyltrimethylammonium p-toluene sulfonate (CTMA) (pH 5.2). Most importantly, the sensitivity of SSC under the optimum separation conditions is not sacrificed. The detection limit is 8 nM (20 microl injected).

Prediction of molecular recognition-enhanced phenobarbital extraction based on solvatochromic analysis.

The extraction efficiency for organic molecules using synthetic receptors is highly dependent on solvent properties. Solvent influences the partitioning of both the desired compound and the interfering species. Solvent also influences the solubility of the receptor and its affinity for the substrate. Therefore the free energy involved in extraction can be optimized by using a carefully selected solvent. In this paper we demonstrate the use of a solvatochromic model to predict the influence of solvent dipolarity, H-bond acidity and H-bond basicity on selectivity and yield of phenobarbital extraction. We also used this method to estimate the purity and yield of phenobarbital extraction in 12 poly(vinyl chloride) plasticizers and solvents. This approach can be generalized for assisting the selection of optimal solvent and provide insight into the rational design of solvent and receptor for industrial extractions.

Bicarbonate-sensitive cysteine induced elevation of extracellular aspartate and glutamate in rat hippocampus in vitro.

The effect of different concentrations of cysteine (0.125, 0.25, 0.5 and 1 mM) on the net efflux of endogenous amino acids was studied by the incubation of rat hippocampal slices. Addition of cysteine (1 mM) in bicarbonate containing low K+ medium (5 min) selectively increased the basal net efflux of glutamate and aspartate by 370% and 396%, respectively. High K+ media (50 mM) containing cysteine (1 mM) evoked the net efflux of glutamate and aspartate by 1454% and 1019%, respectively. The corresponding effects in control slices without cysteine were 669% and 404%, respectively. No changes were observed on the concentrations of GABA, glutamine and taurine. The cysteine oxidation products, cysteine sulfinate (0.5 microM) and cystine (0.25 mM) were without effects. The effect of cysteine (0.5 mM) was dramatically reduced in media with no added bicarbonate/CO2. Thus, cysteine in a bicarbonate-sensitive manner selectively increases the extracellular concentration of excitotoxic amino acids in adult rat brain in vitro, possibly by interfering with the carrier-mediated glutamate uptake/release.

Solvatochromic Study of Poly(vinyl chloride) Plasticizers and Their Solutions in Chloroform: Application to Phenobarbital Partitioning and Molecular Recognition of Phenobarbital.

The possibility now exists, with the availability of several families of artificial molecular receptors, to create selective extraction media. More selective extractions will lead to cleaner chromatograms, with lower detection limits and perhaps higher accuracy for trace organic analysis by chromatography. Furthermore, laboratories will be expected to minimize the use of volatile organic solvents. Consequently, nonvolatile, reusable solvents will be the basis for extractions. In addition, as artificial molecular receptors become more widely available, these solvents will be used to support molecular recognition. We have focused on plasticizers of poly(vinyl chloride) as examples of these solvents. We have determined solvatochromic parameters of several plasticizers and their solutions in chloroform. These parameters, along with cohesive energy density and solvent molar volume, were used to derive linear free energy relationships for the free energies of phenobarbital partitioning between solvent and aqueous solution, receptor solubility, formation of a complex with a barbiturate receptor [1,3-bis[[[6-(1-butyrylamino)pyrid-2-yl]amino]carbonyl]benzene (2)], and the transfer of the complex (artificial receptor and phenobarbital) from chloroform to other solvents. Solvent dipolarity/polarizability and hydrogen bond basicity, but not acidity, support complex dissociation. Solvents with large molar volumes dissolve the polar solutes, phenobarbital, receptor, and complex more poorly than solvents with lower molar volume, but there is no influence of molar volume on complex formation.

Molecular recognition of phenobarbital in plasticizers equilibrium investigations on the solubility of the barbiturate artificial receptor and its binding to phenobarbital in plasticizers.

Environmental concern is renewing interest in selective, waste-free extractions. A recent report demonstrated an improved extraction of phenobarbital by means of a specifically designed molecular receptor. In that work, the solvent was CHCl3. The current work is the first step in extending extractions based on molecular recognition to reusable solvents, namely plasticizers. Phenobarbital aqueous/organic partition coefficients, receptor solubility, and phenobarbital-receptor-formation constants in several plasticizers and in their CHCl3 solutions are reported. In addition, by a thermodynamic cycle, the free energy for transfer of the barbiturate-receptor complex from CHCl3 to plasticizers has been calculated. Finally, the data have been displayed in coordinate systems representing extraction efficiency and selectivity. The most selective extraction medium yielding useful extraction efficiency is dioctyl phthalate.

Determination of the pharmaceutical peptide TP9201 by post-column reaction with copper(II) followed by electrochemical detection.

An electrochemical detection method was applied to the determination of the synthetic peptide TP9201 (Telios Pharmaceuticals). The method utilizes reversed-phase HPLC, followed by post-column formation of Cu(II)-peptide complexes to render peptides electrochemically active via the Cu(III/II) couple. TP9201 is cyclic and N-amidated; the lack of a free amine precludes the use of typical fluorescent labeling reagents. Neither the cyclic structure nor the N-amidation prevented the copper complexation reaction, however. The detection limit in bovine serum was 20 nM, limited by interfering sample peaks, and the detector response was linear in a range 10-400 nM.