Biochemical indicators of implantation success of tissue-engineered oral mucosa.

Real-time (RT) determination of the health of in vitro tissue-engineered constructs prior to grafting is essential for prediction of success of the implanted tissue-engineered graft. In addition, the US Food and Drug Administration requires specific release criteria in RT prior to the release of tissue-engineered devices for human use. In principle, assessing the viability and functionality of the cellular component can be achieved by quantifying the secretion of growth factors and chemokines of tissue-engineered constructs. Ex vivo-produced oral mucosa equivalents (EVPOMEs) were fabricated under thermally stressed conditions at 43 °C for 24 h to create a functionally compromised EVPOME. We used microchannel enzyme-linked immunosorbent assay to evaluate the functionality of the cellular component, oral keratinocytes, of stressed and unstressed EVPOMEs by measuring the release of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), human ß-defensin 1 (hBD-1), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) into the spent medium, which was collected on the same day prior to graft implantation into severe combined immunodeficiency mice. Implanted EVPOMEs' histology on the seventh postimplantation day was used to correlate outcomes of grafting to secreted amounts of IL-8, hBD-1, VEGF, TIMP-1, and TIMP-2 from corresponding EVPOMEs. Our findings showed that significantly higher levels of IL-8, hBD-1, and TIMP-2 were secreted from controls than from thermally stressed EVPOMEs. We also found a direct correlation between secreted VEGF and IL-8 and blood vessel counts of implanted EVPOMEs. We concluded that measuring the constitutive release of these factors can be used as noninvasive predictors of healthy tissue-engineered EVPOMEs in RT, prior to their implantation.

The rate of intravenous cocaine administration alters c-fos mRNA expression and the temporal dynamics of dopamine, but not glutamate, overflow in the striatum.

The rapid entry of drugs into the brain is thought to increase the propensity for addiction. The mechanisms that underlie this effect are not known, but variation in the rate of intravenous cocaine delivery does influence its ability to induce immediate early gene expression (IEG) in the striatum, and to produce psychomotor sensitization. Both IEG induction and psychomotor sensitization are dependent upon dopamine and glutamate neurotransmission within the striatum. We hypothesized, therefore, that varying the rate of intravenous cocaine delivery might influence dopamine and/or glutamate overflow in the striatum. To test this we used microdialysis coupled to on-line capillary electrophoresis and laser-induced fluorescence, which allows for very rapid sampling, to compare the effects of a rapid (5 s) versus a slow (100 s) intravenous cocaine infusion on extracellular dopamine and glutamate levels in the striatum of freely moving rats. An acute injection of cocaine had no effect on extracellular glutamate, at either rate tested. In contrast, although peak levels of dopamine were unaffected by infusion rate, dopamine levels increased more rapidly when cocaine was administered over 5 versus 100 s. Moreover, c-fos mRNA expression in the region of the striatum sampled was greater when cocaine was administered rapidly than when given slowly. These data suggest that small differences in the temporal dynamics of dopamine neurotransmission may have a large effect on the subsequent induction of intracellular signalling cascades that lead to immediate early gene expression, and in this way influence the ability of cocaine to produce long-lasting changes in brain and behavior.

Up-regulation of GLT1 expression increases glutamate uptake and attenuates the Huntington's disease phenotype in the R6/2 mouse.

The striatum, which processes cortical information for behavioral output, is a key target of Huntington's disease (HD), an autosomal dominant condition characterized by cognitive decline and progressive loss of motor control. Increasing evidence implicates deficient glutamate uptake caused by a down-regulation of GLT1, the primary astroglial glutamate transporter. To test this hypothesis, we administered ceftriaxone, a beta-lactam antibiotic known to elevate GLT1 expression (200 mg/kg, i.p., for 5 days), to symptomatic R6/2 mice, a widely studied transgenic model of HD. Relative to vehicle, ceftriaxone attenuated several HD behavioral signs: paw clasping and twitching were reduced, while motor flexibility, as measured in a plus maze, and open-field climbing were increased. Assessment of GLT1 expression in striatum confirmed a ceftriaxone-induced increase relative to vehicle. To determine if the change in behavior and GLT1 expression represented a change in striatal glutamate handling, separate groups of behaving mice were evaluated with no-net-flux microdialysis. Vehicle treatment revealed a glutamate uptake deficit in R6/2 mice relative to wild-type controls that was reversed by ceftriaxone. Vehicle-treated animals, however, did not differ in GLT1 expression, suggesting that the glutamate uptake deficit in R6/2 mice reflects dysfunctional rather than missing GLT1. Our results indicate that impaired glutamate uptake is a major factor underlying HD pathophysiology and symptomology. The glutamate uptake deficit, moreover, is present in symptomatic HD mice and reversal of this deficit by up-regulating the functional expression of GLT1 with ceftriaxone attenuates the HD phenotype.

Differential increase in taurine levels by low-dose ethanol in the dorsal and ventral striatum revealed by microdialysis with on-line capillary electrophoresis.

Ethanol increases taurine efflux in the nucleus accumbens or ventral striatum (VS), a dopaminergic terminal region involved in positive reinforcement. However, this has been found only at ethanol doses above 1 g/kg intraperitoneally, which is higher than what most rats will self-administer. We used a sensitive on-line assay of microdialysate content to test whether lower doses of ethanol selectively increase taurine efflux in VS as opposed to other dopaminergic regions not involved in reinforcement (e.g., dorsal striatum; DS). Adult male rats with microdialysis probes in VS or DS were injected with ethanol (0, 0.5, 1, and 2 g/kg intraperitoneally), and the amino acid content of the dialysate was measured every 11 sec using capillary electrophoresis and laser-induced fluorescence detection. In VS, 0.5 g/kg ethanol significantly increased taurine levels by 20% for 10 min. A similar increase was seen after 1 g/kg ethanol, which lasted for about 20 min after injection. A two-phased taurine efflux was observed with the 2.0 g/kg dose, where taurine was increased by 2-fold after 5 min but it remained elevated by 30% for at least 60 min. In contrast, DS exhibited much smaller dose-related increases in taurine. Glycine, glutamate, serine, and gamma-aminobutyric acid were not systematically affected by lower doses of ethanol; however, 2 g/kg slowly decreased these amino acids in both brain regions during the hour after injection. These data implicate a possible role of taurine in the mechanism of action of ethanol in the VS. The high sensitivity and time resolution afforded by capillary electrophoresis and laser-induced fluorescence detection will be useful for detecting subtle changes of neuronally active amino acids levels due to low doses of ethanol.

Capillary liquid chromatography of multiple peptides with on-line capillary electrophoresis immunoassay detection.

A competitive immunoassay for neuropeptide Y (NPY) based on capillary electrophoresis (CE) with laser-induced fluorescence detection was developed utilizing polyclonal antisera as the immunoreagent and fluorescein-labeled NPY as the tracer. The assay was performed with on-line mixing of reagents, automated injections, and a 3 s separation time. The assay had a detection limit of 850 pM. To detect NPY at lower concentrations, the assay was coupled on-line to reversed-phase capillary liquid chromatography (LC). In this arrangement, 5 microL samples were preconcentrated by capillary LC and eluted by a gradient of isopropanol-containing mobile phase. The resulting chromatographic peaks were monitored by the CE immunoassay. With preconcentration, the concentration detection limit was improved to 40 microM and NPY could be measured in push-pull perfusion samples collected from the paraventricular nucleus of freely moving rats. The technique was extended to simultaneous detection of NPY and glucagon secretion from islets of Langerhans.

In vivo monitoring of amine neurotransmitters using microdialysis with on-line capillary electrophoresis.

Microdialysis sampling was coupled via a flow-gated interface on-line to capillary electrophoresis with laser-induced fluorescence (LIF) detection for in vivo monitoring of neuroactive amino acids and amines. In the instrument, analytes are derivatized precolumn with o-phthaldehyde and beta-mercaptoethanol to form fluorescent isoindole products. The instrument was improved over previous designs by incorporating a sheath-flow cuvette for reduced background in LIF detection which improved sensitivity by 15-fold. The methodology was improved by utilizing a voltage ramped injection which allowed generation of 500000 theoretical plates with 20 s separations. Resolution of the isoindole derivatives was further improved by addition of hydroxypropyl-modified beta-cyclodextrin to the electrophoresis buffer. The new instrumentation and methods allow resolution and detection of glutamate, gamma-aminobutyric acid, glycine, aspartate, serine, taurine, glutamine and dopamine (if levels are elevated) collected from in vivo sampling probes every 20 s. The technique is suited to continuous monitoring for dynamic measurements of these compounds in vivo.

Capillary LC-MS2 at the attomole level for monitoring and discovering endogenous peptides in microdialysis samples collected in vivo.

Fused-silica capillary LC columns (25-microm i.d.) with 3-microm-i.d. integrated electrospray emitters interfaced to a quadrupole ion trap mass spectrometer were evaluated for high-sensitivity LC-MS2. Column preparation involved constructing frits by in situ photopolymerization of glycidyl methacrylate and trimethylolpropane trimethacrylate, preparing the electrospray emitter by pulling the column outlet to a fine tip with a CO2 laser puller, and slurry-packing the column with 5-microm reversed-phase particles. Large-volume injections were facilitated by an automated two-pump system that allowed high-flow rates for sample loading and low-flow rates for elution. Small electrospray emitters, low elution flow rates, and optimization of gradient steepness allowed a detection limit of 4 amol, corresponding to 2 pM for 1.8 microL injected on-column, for a mixture of peptides dissolved in artificial cerebral spinal fluid. The system was coupled on-line to microdialysis sampling and was used to monitor and discover endogenous neuropeptides from the globus pallidus of anesthetized male Sprague-Dawley rats. Time-segmented MS2 scans enabled simultaneous monitoring of Met-enkephalin, Leu-enkephalin, and unknown peptides. Basal dialysate levels of Met-enkephalin and Leu-enkephalin were 60 +/- 30 and 70 +/- 20 pM while K+-stimulated levels were 1,900 +/- 500 and 1,300 +/- 300 pM, respectively (n = 7). Data-dependent and time-segmented MS2 scans revealed several unknown peptides that were present in dialysate. One of the unknowns was identified as peptide I(1-10) (SPQLEDEAKE), a novel product of preproenkephalin A processing, using MS2, MS3, and database searching.

Spatial organization of Ca(2+) entry and exocytosis in mouse pancreatic beta-cells.

Secretion from single pancreatic beta-cells was imaged using a novel technique in which Zn(2+), costored in secretory granules with insulin, was detected by confocal fluorescence microscopy as it was released from the cells. Using this technique, it was observed that secretion from beta-cells was limited to an active region that comprised approximately 50% of the cell perimeter. Using ratiometric imaging with indo-1, localized increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by membrane depolarization were also observed. Using sequential measurements of secretion and [Ca(2+)](i) at single cells, colocalization of exocytotic release sites and Ca(2+) entry was observed when cells were stimulated by glucose or K(+). Treatment of cells with the Ca(2+) ionophore 4-Br-A23187 induced large Ca(2+) influx around the entire cell circumference. Despite the nonlocalized increase in [Ca(2+)](i), secretion evoked by 4-Br-A23187 was still localized to the same region as that evoked by secretagogues such as glucose. It is concluded that Ca(2+) channels activated by depolarization are localized to specific membrane domains where exocytotic release also occurs; however, localized secretion is not exclusively regulated by localized increases in [Ca(2+)](i), but instead involves spatial localization of other components of the exocytotic machinery.

GABA(B) receptors mediate motility signals for migrating embryonic cortical cells.

During development, postmitotic neurons migrate from germinal regions into the cortical plate (cp), where lamination occurs. In rats, GABA is transiently expressed in the cp, near target destinations for migrating neurons. In vitro GABA stimulates neuronal motility, suggesting cp cells release GABA, which acts as a chemoattractant during corticogenesis. Pharmacological studies indicate GABA stimulates migration via GABA(B)-receptor (GABA(B)-R) activation. Using immunohistochemistry, RT-PCR and Western blotting, we examined embryonic cortical cell expression of GABA(B)-Rs in vivo. At E17, GABA(B)-R1(+) cells were identified in the ventricular zone (vz) and cp. RT-PCR and Western blotting demonstrated the presence of GABA(B)-R1a and GABA(B)-R1b mRNA and proteins. Using immuno- cytochemistry, GABA(B)-R expression was examined in vz and cp cell dissociates before and after migration to GABA in an in vitro chemotaxis assay. GABA-induced migration resulted in an increase of GABA(B)-R(+) cells in the migrated population. While <20% of each starting dissociate was GABA(B)-R(+), >70% of migrated cells were immunopositive. We used a microchemotaxis assay to analyze cp cell release of diffusible chemotropic factor(s). In vitro, cp dissociates induced vz cell migration in a cell density-dependent manner that was blocked by micromolar saclofen (a GABA(B)-R antagonist). HPLC demonstrated cp cells release micromolar levels of GABA and taurine in several hours. Micromolar levels of both molecules stimulated cell migration that was blocked by micromolar saclofen. Thus, migratory cortical cells express GABA(B)-Rs, cp cells release GABA and taurine, and both molecules stimulate cortical cell movement. Together these findings suggest GABA and/or taurine act as chemoattractants for neurons during rat cortical histogenesis via mechanisms involving GABA(B)-Rs.

Retention and separation of adenosine and analogues by affinity chromatography with an aptamer stationary phase.

A biotinylated-DNA aptamer (molecular weight 16,600) that binds adenosine and related compounds in solution was immobilized by reaction with streptavidin, which had been covalently attached to porous chromatographic supports. The aptamer medium was packed into fused-silica capillaries (50-150-microm i.d.) to form affinity chromatography columns. Frontal chromatography analysis indicated that the dissociation constants (Kd) of cyclic-AMP, AMP, ATP, ADP, and adenosine were 138 +/- 18, 58 +/- 2, 38 +/- 2, 28 +/- 6 and 3 +/- 1 microM, respectively, for aptamer immobilized on a controlled pore glass support. Similar values were obtained for aptamer immobilized on a polystyrene support except for a slightly higher Kd for adenosine. The Kd for adenosine is similar to the previously reported value of 6 +/- 3 microM for adenosine-aptamer in solution indicating that immobilized aptamers can have affinity similar to that of the solution forms. Columns had 20 nmol of binding sites/100 microL of support media, which is 3.3-fold higher than that previously reported for immobilization of IgG on similar media, indicating that the aptamer can be immobilized with higher density than antibodies. Variation of mobile-phase conditions revealed that ionic strength and Mg2+ level had strong effects on retention of analytes while pH and buffer composition had less of an effect. It was demonstrated that the column could selectively retain and separate cyclic-AMP, NAD+, AMP, ADP, ATP, and adenosine, even in complex mixtures such as tissue extracts.

Reversed-phase capillary liquid chromatography coupled on-line to capillary electrophoresis immunoassays.

Capillary reversed-phase liquid chromatography (RPLC) was coupled on-line to competitive capillary electrophoresis immunoassay (CEIA) to improve concentration sensitivity of the competitive CEIA and to provide a means for detecting multiple species that cross-react with antibody. A competitive CEIA for glucagon was used for demonstration of this technique. Five-microliter samples were injected onto a 4-cm-long by 50-micron-i.d. RPLC column. Sample was desorbed by gradient elution, mixed on-line with fluorescently labeled glucagon and anti-glucagon, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injection and laser-induced fluorescence detection. Electrophoretic analysis of the reactor stream was performed every 1.5 s, allowing nearly continuous monitoring of the RPLC separation. Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 to 20 pM. Addition of the RPLC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay. The technique was used to measure glucagon secretion from single islets of Langerhans and to differentiate cross-reactive forms of glucagon with one assay.

Rapid simultaneous determination of glucagon and insulin by capillary electrophoresis immunoassays.

A rapid capillary electrophoresis (CE) with laser-induced fluorescence (LIF) competitive immunoassay has been developed for the determination of glucagon in biological mixtures. In the assay, fluorescein-conjugated glucagon is mixed with the sample followed by addition of anti-glucagon. Free and antibody-bound, tagged glucagon could be separated in 3 s using CE to obtain quantitative determination of glucagon with a concentration detection limit of 760 pM. The assay was combined with a previously developed competitive immunoassay for insulin to produce a simultaneous immunoassay for both peptides. The method was used to determine glucagon content of islets of Langerhans.

Roles of insulin receptor substrate-1, phosphatidylinositol 3-kinase, and release of intracellular Ca2+ stores in insulin-stimulated insulin secretion in beta -cells.

The signaling pathway by which insulin stimulates insulin secretion and increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in isolated mouse pancreatic beta-cells and clonal beta-cells was investigated. Application of insulin to single beta-cells resulted in increases in [Ca(2+)](i) that were of lower magnitude, slower onset, and longer lifetime than that observed with stimulation with tolbutamide. Furthermore, the increases in [Ca(2+)](i) originated from interior regions of the cell rather than from the plasma membrane as with depolarizing stimuli. The insulin-induced [Ca(2+)](i) changes and insulin secretion at single beta-cells were abolished by treatment with 100 nm wortmannin or 1 micrometer thapsigargin; however, they were unaffected by 10 micrometer U73122, 20 micrometer nifedipine, or removal of Ca(2+) from the medium. Insulin-stimulated insulin secretion was also abolished by treatment with 2 micrometer bisindolylmaleimide I, but [Ca(2+)](i) changes were unaffected. In an insulin receptor substrate-1 gene disrupted beta-cell tumor line, insulin did not evoke either [Ca(2+)](i) changes or insulin secretion. The data suggest that autocrine-activated increases in [Ca(2+)](i) are due to release of intracellular Ca(2+) stores, especially the endoplasmic reticulum, mediated by insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Autocrine activation of insulin secretion is mediated by the increase in [Ca(2+)](i) and activation of protein kinase C.

Detection of secretion from single pancreatic beta-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy.

Confocal microscopy with Zinquin, a fluorogenic Zn(2+)-specific indicator, was used for spatially and temporally resolved measurement of Zn2+ efflux from single pancreatic beta-cells. When cells were incubated in buffer containing Zinquin, application of insulin secretagogues evoked an increase in fluorescence around the surface of the cell, indicative of detection of Zn2+ efflux from the cell. The fluorescence increases corresponded spatially and temporally with measurements of exocytosis obtained simultaneously by amperometry. When images were taken at 266-ms intervals, the detection limit for Zn2+ was approximately 0.5 microM. With this image frequency, it was possible to observe bursts of fluorescence which were interpreted as fluctuations of Zn2+ level due to exocytosis. The average intensity of these fluorescence bursts corresponded to a Zn2+ concentration of approximately 7 microM. Since insulin is co-stored with Zn2+ in secretory vesicles, it was concluded that the Zn2+ efflux corresponded to exocytosis of insulin/Zn(2+)-containing granules from the beta-cell. Exocytosis sites identified by this technique were frequently localized to one portion of the cell, indicative of active areas of release.

Trace-level amino acid analysis by capillary liquid chromatography and application to in vivo microdialysis sampling with 10-s temporal resolution.

A sensitive method was developed to determine 16 amino acids, including all the neurotransmitter amino acids and neuromodulators, in physiological samples. Samples were derivatized with o-phthalaldehyde/tert-butyl thiol followed by two scavenging reactions that reduced the chemical background caused by excess derivatization reagent by approximately 90%. A total of 250 nL of the derivatized sample was injected and concentrated onto a 50-micron-inner diameter capillary column packed with 5-micron reversed-phase particles and separated using gradient elution. Analytes were detected amperometrically at a cylindrical 9-micron carbon fiber microelectrode. The combination of on-column concentration, scavenging reactions after derivatization, high sensitivity electrochemical detection, and protocols to minimize amine contamination allowed detection limits of 90-350 pM (20-80 amol) for all the amino acids tested. This method was used to analyze in vivo microdialysate samples from probes implanted in the striatum of anesthetized rats. Probes were perfused at 1.2 microL/min and fractions collected every 10 s. The 200-nL fractions were diluted to 2 microL to facilitate sample handling for off-line analysis. The suitability of this method for simultaneous monitoring of all the major amino acid neurotransmitters with 10-s temporal resolution under basal conditions, during potassium stimulation, and during selective uptake inhibition of gamma-aminobutyric acid is demonstrated.

Correlated oscillations in glucose consumption, oxygen consumption, and intracellular free Ca(2+) in single islets of Langerhans.

Micron-sized sensors were used to monitor glucose and oxygen levels in the extracellular space of single islets of Langerhans in real-time. At 10 mM glucose, oscillations in intraislet glucose concentration were readily detected. Changes in glucose level correspond to changes in glucose consumption by glycolysis balanced by mass transport into the islet. Oscillations had a period of 3.1 +/- 0.2 min and amplitude of 0.8 +/- 0.1 mM glucose (n = 21). Superimposed on these oscillations were faster fluctuations in glucose level during the periods of low glucose consumption. Oxygen level oscillations that were out of phase with the glucose oscillations were also detected. Oscillations in both oxygen and glucose consumption were strongly dependent upon extracellular Ca(2+) and sensitive to nifedipine. Simultaneous measurements of glucose with intracellular Ca(2+) ([Ca(2+)](i)) revealed that decreases in [Ca(2+)](i) preceded increases in glucose consumption by 7.4 +/- 2.1 s during an oscillation (n = 9). Conversely, increases in [Ca(2+)](i) preceded increases in oxygen consumption by 1.5 +/- 0.2 s (n = 4). These results suggest that during oscillations, bursts of glycolysis begin after Ca(2+) has stopped entering the cell. Glycolysis stimulates further Ca(2+) entry, which in turn stimulates increases in respiration. The data during oscillation are in contrast to the time course of events during initial exposure to glucose. Under these conditions, a burst of oxygen consumption precedes the initial rise in [Ca(2+)](i). A model to explain these results is described.

Localized exocytosis detected by spatially resolved amperometry in single pancreatic beta-cells.

Spatially resolved measurements of exocytosis in pancreatic beta-cells were made using amperometry with 1-microm radius electrodes. These measurements revealed that certain portions of a cell actively undergo exocytosis following stimulation with depolarizing agents, but other regions are inactive. The amperometric measurements were compared to measurements made with the membrane indicator dye, FM1-43, which showed uneven increases in fluorescence around the surface of the cell, with amperometric secretion being detected only at the brightest regions. In some instances, a large number of exocytotic events were detected from one electrode position. The number of events was larger than what would be expected based on the number of vesicles that could fit under an electrode of the dimensions used. These results suggest a mechanism of vesicle traffic that allows multiple fusions at a small membrane area.

Identification, quantitation, and characterization of biomolecules by capillary electrophoretic analysis of binding interactions.

The high resolving power of capillary electrophoresis combined with the specificity of binding interactions may be used with advantage to characterize the structure-function relationship of biomolecules, to quantitate specific analytes in complex sample matrices, and to determine the purity of pharmaceutical and other molecules. We here review recent and innovative methodologies and applications of high resolution affinity electrophoresis within the fields of binding constant determination, structure-activity studies, quantitative microassays, analysis of drug purity and protein conformation, and immobilized affinity ligands. Despite the virtues of these approaches with respect to applicability, resolving power, speed, and low sample consumption, problems remain with respect to analyte identification and low concentration limits of detection. The ongoing development of new detector technologies for capillary electrophoresis such as mass spectrometry, and possibly nuclear magnetic resonance and other spectroscopic methods, is therefore very promising for the continued increased use of affinity capillary electrophoresis.

Oxygen microsensor and its application to single cells and mouse pancreatic islets.

An oxygen microsensor with a < 3-micron tip diameter was developed for monitoring oxygen levels at single cells and mouse pancreatic islets. The sensor was fabricated by electrochemically recessing an etched Pt wire inside a pulled glass micropipet and then coating with cellulose acetate. This fabrication process was found to be simpler than previous oxygen electrode designs of comparable size. The microsensors had a average sensitivity of 0.59 +/- 0.29 pA/mmHg (mean +/- SD, n = 42), signals that were minimally perturbed by convection, and response times of < 1 s. The electrode was used to measure the oxygen gradient around and inside single mouse islets. The measurements demonstrate that oxygen levels within even the largest islets at maximal glucose stimulation are 67 +/- 1.6 mmHg (mean +/- SD, n = 5), indicating that islets have adequate oxygen supplies by diffusion under tissue culture conditions to support insulin secretion. The electrode was also used to record the dynamics of oxygen level at single islets as a function of glucose concentration. As glucose level was changed from 3 to 10 mM, oxygen level decreased by 15.8 +/- 2.3 mmHg (mean +/- SEM, n = 6) and oscillations with a period of 3.3 +/- 0.6 min (mean +/- SEM, n = 6) appeared in the oxygen level. In islets bathed in quiescent solutions containing 10 mM glucose, similar oscillations could be observed. In addition, in the quiet solutions it was possible to detect faster oscillations with a period of 12.1 +/- 1.7 s (mean +/- SEM, n = 6) superimposed on the slower oscillations. Oxygen consumption could also be observed at single insulinoma cells using the electrode. Individual cells also showed oscillations in oxygen consumption with a period of a few seconds. The results demonstrate that the electrode can be used for dynamic oxygen level recordings in biological microenvironments.

Rapid determination of aspartate enantiomers in tissue samples by microdialysis coupled on-line with capillary electrophoresis.

Microdialysis was coupled on-line with derivatization by o-phthalaldehyde and beta-mercaptoethanol and optically gated capillary electrophoresis to determine D- and L-aspartate in tissue samples obtained from rats. The microdialysis probe was inserted into a homogenized tissue sample which allowed generation of a continuous sample stream that was filtered and deproteinated. With 7.5 mM beta-cyclodextrin (CD) in the electrophoresis buffer, the enantiomers of interest could be resolved in 3 s with an electric field of 2500 V/cm over a separation length of 15 mm. Values of D- and L-aspartate in different tissues agreed well with those obtained by an HPLC procedure that required protein precipitation, centrifugation, and extraction. The speed and compatibility with automation of the microdialysis/CE method may make it a general approach for a variety of applications involving high-throughput analysis or sensorlike operation.