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Objective Post–bariatric surgery hypoglycemia (PBH) is defined as the presence of neuroglycopenic symptoms accompanied by postprandial hypoglycemia in bariatric surgery patients. Recent clinical studies using continuous glucose monitoring (CGM) technology revealed that PBH is more frequently observed in vertical sleeve gastrectomy (VSG) patients than previously recognized. PBH cannot be alleviated by current medication. Therefore, a model system to investigate the mechanism and treatment is required. Methods We used CGM in a rat model of VSG and monitored the occurrence of glycemic variability and hypoglycemia in various meal conditions for 4 weeks after surgery. Another cohort of VSG rats with CGM was used to investigate whether the blockade of glucagon-like peptide-1 receptor (GLP-1R) signaling alleviates these symptoms. A mouse VSG model was used to investigate whether the impaired glucose counterregulatory system causes postprandial hypoglycemia. Results Like in humans, rats have increased glycemic variability and hypoglycemia after VSG. Postprandial hypoglycemia was specifically detected after liquid versus solid meals. Further, the blockade of GLP-1R signaling raises the glucose nadir but does not affect glycemic variability. Conclusions Rat bariatric surgery duplicates many features of human post–bariatric surgery hypoglycemia including postprandial hypoglycemia and glycemic variability, while blockade of GLP-1R signaling prevents hypoglycemia but not the variability., Highlights • VSG causes glycemic variability during ad lib feeding condition. • Single liquid meal ingestion causes post-VSG hypoglycemia. • Blockade of GLP-1 receptor prevents post-VSG hypoglycemia.

Comprehensive characterization of the lipidome remains a challenge requiring development of new analytical approaches to expand lipid coverage in complex samples. In this work, offline two-dimensional liquid chromatography-mass spectrometry was investigated for lipidomics from human plasma. Hydrophilic interaction liquid chromatography was implemented in the first dimension to fractionate lipid classes. Nine fractions were collected and subjected to a second-dimension separation utilizing 50 cm capillary columns packed with 1.7 µm C18 particles operated on custom-built instrumentation at 35 kpsi. Online coupling with time-of-flight mass spectrometry allowed putative lipid identification from precursor-mass based library searching. The method had good orthogonality (fractional coverage of ∼40%), achieved a peak capacity of approximately 1900 in 600 min, and detected over 1000 lipids from a 5 µL injection of a human plasma extract while consuming less than 3 mL of solvent. The results demonstrate the expected gains in peak capacity when employing long columns and two-dimensional separations and illustrate practical approaches for improving lipidome coverage from complex biological samples.

Traditional Western blots are commonly used to separate and assay proteins; however, they have limitations including a long, cumbersome process and large sample requirements. Here, we describe a system for Western blotting where capillary gel electrophoresis is used to separate sodium dodecyl sulfate-protein complexes. The capillary outlet is threaded into a piezoelectric inkjetting head that deposits the separated proteins in a quasi-continuous stream of100 pL droplets onto a moving membrane. Through separations at 400 V/cm and protein capture on a membrane moving at 2 mm/min, we are able to detect actin with a limit of detection at 8 pM, or an estimated 5 fg injected. Separation and membrane capture of sample containing 10 proteins ranging in molecular weights from 11 - 250 kDa was achieved in 15 min. The system was demonstrated with Western blots for actin, β-tubulin, ERK1/2, and STAT3 in human A431 epidermoid carcinoma cell lysate.

Previous studies have demonstrated a role for norepinephrine (NE) in energy regulation and feeding, and basal differences have been observed in hypothalamic NE systems in obesity-prone vs. obesity-resistant rats. Differences in the function of brain reward circuits, including in the nucleus accumbens (NAc), have been shown in obesity-prone vs. obesity-resistant populations, leading many researchers to explore the role of striatal dopamine in obesity. However, alterations in NE transmission also affect NAc mediated behaviors. Therefore, here we examined differences in striatal NE and the response to norepinephrine transporter blockers in obesity-prone and obesity-resistant rats. We found that striatal NE levels increase following systemic cocaine administration in obesity-prone, but not obesity-resistant rats. This could result from either blockade of striatal norepinephrine transporters (NET) by cocaine leading to reduced NE reuptake, or circuit-based responses following cocaine administration resulting in increased NE release. Retrodialysis of the NET inhibitor, desipramine, into the ventral striatum did not cause selective increases in striatal NE levels in obesity-prone rats, suggesting that circuit-based mechanisms underlie NE increases following systemic cocaine administration. Consistent with this, systemic desipramine treatment decreased locomotor activity in obesity-prone, but not obesity-resistant rats. Furthermore, obesity-prone rats were also more sensitive to desipramine-induced reductions in food intake compared to obesity-resistant rats. Taken together, these data expand our understanding of differences in NE systems of obesity-prone vs. resistant rats, and provide new insights into basal differences in striatal systems that may influence feeding behavior.

Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (Virus Inhibitory Protein, Endoplasmic Reticulum-associated, Interferon iNducible) is an endoplasmic reticulum membrane-associated enzyme that exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. However, the relationship between viperin expression and the retarded budding of virus particles from lipid rafts on the cell membrane is unclear. Here, we investigated the effect of viperin expression on cholesterol biosynthesis using transiently expressed genes in the human cell line human embryonic kidney 293T (HEK293T). We found that viperin expression reduces cholesterol levels by 20% to 30% in these cells. Following this observation, a proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits, including lanosterol synthase (LS) and squalene monooxygenase (SM), which are enzymes that catalyze key steps in establishing the sterol carbon skeleton. Coimmunoprecipitation experiments confirmed that viperin, LS, and SM form a complex at the endoplasmic reticulum membrane. While coexpression of viperin was found to significantly inhibit the specific activity of LS in HEK293T cell lysates, coexpression of viperin had no effect on the specific activity of SM, although did reduce SM protein levels by approximately 30%. Despite these inhibitory effects, the coexpression of neither LS nor SM was able to reverse the viperin-induced depletion of cellular cholesterol levels, possibly because viperin is highly expressed in transfected HEK293T cells. Our results establish a link between viperin expression and downregulation of cholesterol biosynthesis that helps explain viperin's antiviral effects against enveloped viruses.

Two separate liquid chromatography (LC)-mass spectrometry (MS) methods were developed for determination and quantification of water-soluble and fat-soluble vitamins in human tear and blood serum samples. The water-soluble vitamin method was originally developed to detect vitamins B1, B2, B3 (nicotinamide), B5, B6 (pyridoxine), B7, B9 and B12 while the fat-soluble vitamin method detected vitamins A, D3, 25(OH)D3, E and K1. These methods were then validated with tear and blood serum samples. In this data in brief article, we provide details on the two LC-MS methods development, methods sensitivity, as well as precision and accuracy for determination of vitamins in human tears and blood serum. These methods were then used to determine the vitamin concentrations in infant and parent samples under a clinical study which were reported in "Determination of Water-Soluble and Fat-Soluble Vitamins in Tears and Blood Serum of Infants and Parents by Liquid Chromatography/Mass Spectrometry DOI:10.1016/j.exer.2016.12.007 [1]". This article provides more details on comparison of vitamin concentrations in the samples with the ranges reported in the literature along with the medically accepted normal ranges. The details on concentrations below the limits of detection (LOD) and limits of quantification (LOQ) are also discussed. Vitamin concentrations were also compared and cross-correlated with clinical data and nutritional information. Significant differences and strongly correlated data were reported in [1]. This article provides comprehensive details on the data with slight differences or slight correlations.

Tears serve as a viable diagnostic fluid with advantages including less invasive sample to collect and less complex to prepare for analysis. Several water-soluble and fat-soluble vitamins were detected and quantified in human tears and compared with blood serum levels. Samples from 15 family pairs, each pair consisting of a four-month-old infant and one parent were analyzed; vitamin concentrations were compared between tears and blood serum for individual subjects, between infants and parents, and against self-reported dietary intakes. Water-soluble vitamins B1, B2, B3 (nicotinamide), B5, B9 and fat-soluble vitamin E (α-tocopherol) were routinely detected in tears and blood serum while fat-soluble vitamin A (retinol) was detected only in blood serum. Water-soluble vitamin concentrations measured in tears and blood serum of single subjects were comparable, while higher concentrations were measured in infants compared to their parents. Fat-soluble vitamin E concentrations were lower in tears than blood serum with no significant difference between infants and parents. Serum vitamin A concentrations were higher in parents than infants. Population trends were compiled and quantified using a cross correlation factor. Strong positive correlations were found between tear and blood serum concentrations of vitamin E from infants and parents and vitamin B3 concentrations from parents, while slight positive correlations were detected for infants B3 and parents B1 and B2 concentrations. Correlations between infants and parents were found for the concentrations of B1, B2, B3, and E in tears, and the concentrations of B2, A, and E in blood serum. Stronger vitamin concentration correlations were found between infants and parents for the breast-fed infants, while no significant difference was observed between breast-fed and bottle-fed infants. This work is the first to demonstrate simultaneous vitamin A, B, and E detection and to quantify correlations between vitamin concentrations in tears and blood serum. Our results suggest that tears are a viable biofluid to monitor nutritional health because they sufficiently mirror blood serum data and may enhance the speed of deficiency diagnoses.

Objective Glucose-stimulated insulin secretion in pancreatic beta cells requires metabolic signals including the generation of glucose-derived short chain acyl-CoAs in the cytosol from mitochondrially-derived metabolites. One concept of insulin secretion is that ATP citrate lyase generates short chain acyl-CoAs in the cytosol from mitochondrially-derived citrate. Of these, malonyl-CoA, is believed to be an important signal in insulin secretion. Malonyl-CoA is also a precursor for lipids. Our recent evidence suggested that, in the mitochondria of beta cells, glucose-derived pyruvate can be metabolized to acetoacetate that is exported to the cytosol and metabolized to the same short chain acyl-CoAs and fatty acids that can be derived from citrate. We tested for redundancy of the citrate pathway. Methods We inhibited ATP citrate lyase activity using hydroxycitrate as well as studying a stable cell line generated with shRNA knockdown of ATP citrate lyase in the pancreatic beta cell line INS-1 832/13. Results In both instances glucose-stimulated insulin release was not inhibited. Mass spectrometry analysis showed that the flux of carbon from [U-13C]glucose and/or [U-13C]α-ketoisocaproic acid (KIC) into short chain acyl-CoAs in cells with hydroxycitrate-inhibited ATP citrate lyase or in the cell line with stable severe (>90%) shRNA knockdown of ATP citrate lyase was similar to the controls. Both 13C-glucose and 13C-KIC introduced substantial 13C labeling into acetyl-CoA, malonyl-CoA, and HMG-CoA under both conditions. Glucose flux into fatty acids was not affected by ATP citrate lyase knockdown. Conclusion The results establish the involvement of the acetoacetate pathway in insulin secretion in pancreatic beta cells., Highlights • In pancreatic beta cells mitochondria synthesize metabolites from glucose. • Mitochondria-derived citrate and acetoacetate can transfer carbon to the cytosol. • The citrate pathway requires ATP citrate lyase (ACLY). • Inhibition of ACLY did not stop metabolite export to the cytosol or insulin release. • The results establish the role of the acetoacetate pathway in insulin secretion.

The impact of viscous friction on eluent temperature and column efficiency in liquid chromatography is of renewed interest as the need for pressures exceeding 1000 bar to use with columns packed with sub-2 μm particles has grown. One way the development of axial and radial temperature gradients that arise due to viscous friction can be affected is by the thermal environment the column is placed in. In this study, a new column oven integrated into an ultrahigh pressure liquid chromatograph that enables both still-air and forced-air operating modes is investigated to find the magnitude of the effect of the axial thermal gradient that forms in 2.1 × 100 mm columns packed with sub-2 μm particles in these modes. Temperature increases of nearly 30 K were observed when the generated power of the column exceeded 25 W/m. The impact of the heating due to viscous friction on the repeatability of peak capacity, elution time, and peak area ratio to an internal standard for a gradient UHPLC-MS/MS method to analyze neurotransmitters was found to be limited. This result indicates that high speed UHPLC-MS/MS gradient methods under conditions of high viscous friction may be possible without the negative effects typically observed with isocratic separations under similar conditions.

Recently we reported that nucleus accumbens (NAcc) dopamine (DA) tracks uncertainty during operant responding for non-caloric saccharin. We also showed that repeated intermittent exposure to this uncertainty, like exposure to drugs of abuse, leads to sensitization of the locomotor and NAcc DA effects of amphetamine and promotes the subsequent self-administration of the drug. Here we review these findings together with others showing that NAcc glutamate signaling is similarly affected by uncertainty. Extracellular levels of glutamate in this site also track uncertainty in a task in which nose poking for saccharin on an escalating variable ratio schedule of reinforcement is associated with progressively increasing variance between performance of the operant and payout. Furthermore, sensitized behavioral responding to and for amphetamine following exposure to uncertainty is accompanied by increased levels of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) phosphorylation as well as altered protein levels of the transcription factor ΔFosB (increased) and glutamate transporter 1 (GLT1; decreased) in NAcc tissues. Notably, phosphorylation by CaMKII and PKC regulates AMPA receptor trafficking and function in this site, is elevated following psychostimulant exposure, and is necessary for the expression of enhanced drug taking. Increased ΔFosB and decreased GLT1 levels are observed following psychostimulant exposure, are associated with increased drug taking and seeking, and are known to modulate AMPA receptors and extracellular glutamate levels respectively. These adaptations in glutamate transmission as well as those observed with DA following repeated intermittent exposure to uncertainty are similar to those produced by exposure to abused drugs. Together, they point to the recruitment of both DA and glutamate signaling pathways in the NAcc in both drug and behavioral addictions. As uncertainty is central to games of chance, these findings have particular relevance for gambling disorders known to exhibit comorbidity with drug abuse.

Improvements in sample preparation, separation, and mass spectrometry continue to expand the coverage in LC-MS based lipidomics. While longer columns packed with smaller particles in theory give higher separation performance compared to shorter columns, the implementation of this technology above commercial limits has been sparse due to difficulties in packing long columns and successfully operating instruments at ultrahigh pressures. In this work, a liquid chromatograph that operates up to 35 kpsi was investigated for the separation and identification of lipid species from human plasma. Capillary columns between 15–50 cm long were packed with 1.7 µm BEH C18 particles and evaluated for their ability to separate lipid isomers and complex lipid extracts from human plasma. Putative lipid class identifications were assigned using accurate mass and relative retention time data of the eluting peaks. Our findings indicate that longer columns packed and operated at 35 kpsi outperform shorter columns packed and run at lower pressures in terms of peak capacity and numbers of features identified. Packing columns with relatively high concentration slurries (200 mg/mL) while sonicating the column resulted in 6–34% increase in peak capacity for 50 cm columns compared to lower slurry concentrations and no sonication. For a given analysis time, 50 cm long columns operated at 35 kpsi provided a 20–95% increase in chromatographic peak capacity compared with 15 cm columns operated at 15 kpsi. Analysis times up to 4 h were evaluated, generating peak capacities up to 410 ± 5 (n = 3, measured at 4σ) and identifying 480 ± 85 lipids (n = 2). Importantly, the results also show a correlation between the peak capacity and the number of lipids identified from a human plasma extract. This correlation indicates that ionization suppression is a limiting factor in obtaining sufficient signal for identification by mass spectrometry. The result also shows that the higher resolution obtained by shallow gradients overcomes possible signal reduction due to broader, more dilute peaks in long gradients for improving detection of lipids in LC-MS. Lastly, longer columns operated at shallow gradients allowed for the best separation of both regional and geometrical isomers. These results demonstrate a system that enables the advantages of using longer columns packed and run at ultrahigh pressure for improving lipid separations and lipidome coverage.

Acute fatty acid (FA) exposure potentiates glucose-stimulated insulin secretion in β cells through metabolic and receptor-mediated effects. We assessed the effect of fatty acids on the dynamics of the metabolome in INS-1 cells following exposure to [U-(13)C]glucose to assess flux through metabolic pathways. Metabolite profiling showed a fatty acid-induced increase in long chain acyl-CoAs that were rapidly esterified with glucose-derived glycerol-3-phosphate to form lysophosphatidic acid, mono- and diacylglycerols, and other glycerolipids, some implicated in augmenting insulin secretion. Glucose utilization and glycolytic flux increased, along with a reduction in the NADH/NAD(+) ratio, presumably by an increase in conversion of dihydroxyacetone phosphate to glycerol-3-phosphate. The fatty acid-induced increase in glycolysis also resulted in increases in tricarboxylic cycle flux and oxygen consumption. Inhibition of fatty acid activation of FFAR1/GPR40 by an antagonist decreased glycerolipid formation, attenuated fatty acid increases in glucose oxidation, and increased mitochondrial FA flux, as evidenced by increased acylcarnitine levels. Conversely, FFAR1/GPR40 activation in the presence of low FA increased flux into glycerolipids and enhanced glucose oxidation. These results suggest that, by remodeling glucose and lipid metabolism, fatty acid significantly increases the formation of both lipid- and TCA cycle-derived intermediates that augment insulin secretion, increasing our understanding of mechanisms underlying β cell insulin secretion.

Glutamate is known to cause the release of dopamine through a Ca(2+)-sensitive mechanism that involves activation of NMDA ionotropic glutamate receptors. In the current study, we tested the hypothesis that the delta opioid agonist SNC80 acts indirectly, via the glutamatergic system, to enhance both amphetamine-stimulated dopamine efflux from striatal preparations and amphetamine-stimulated locomotor activity. SNC80 increased extracellular glutamate content, which was accompanied by a concurrent decrease in GABA levels. Inhibition of NMDA signaling with the selective antagonist MK801 blocked the enhancement of both amphetamine-induced dopamine efflux and hyperlocomotion observed with SNC80 pretreatment. Addition of exogenous glutamate also potentiated amphetamine-stimulated dopamine efflux in a Mg(2+)- and MK801-sensitive manner. After removal of Mg(2+) to relieve the ion conductance inhibition of NMDA receptors, SNC80 both elicited dopamine release alone and produced a greater enhancement of amphetamine-evoked dopamine efflux. The action of SNC80 to enhance amphetamine-evoked dopamine efflux was mimicked by the GABA(B) antagonist 2-hydroxysaclofen. These cumulative findings suggest SNC80 modulates amphetamine-stimulated dopamine efflux through an intra-striatal mechanism involving inhibition of GABA transmission leading to the local release of glutamate followed by subsequent activation of NMDA receptors.

Western blotting is a highly valued method for protein identification and relative quantitation in complex samples. It combines size-based electrophoretic separation with immunoaffinity to identify specific proteins. This technique remains popular and has become a workhorse in biochemical research and clinical laboratories. Despite its utility and popularity, this method has many limitations including slow analysis, incompatibility with limited sample application, low throughput and low information content. Recently there has been significant success in improving different aspects of Western blotting. In this review, we provide an overview of the developments in the area of improving conventional Western blotting methods with a focus on recent developments in microfluidic Western blotting. We overview different separation platforms, and discuss studies on protein transfer methods as well as protein immobilization methods and chemistries. We also describe integrated miniaturized platforms that can perform rapid separations and immunodetections.

Background It is not clear whether dabigatran is as safe and effective as uninterrupted anticoagulation with warfarin during radiofrequency catheter ablation (RFA) of atrial fibrillation (AF). Objective To compare the safety and efficacy of dabigatran by using a novel administration protocol and uninterrupted anticoagulation with warfarin for periprocedural anticoagulation in patients undergoing RFA of AF. Methods In this case-control analysis, 763 consecutive patients (mean age 61±10 years) underwent RFA of AF using dabigatran (N = 191) or uninterrupted warfarin (N = 572) for periprocedural anticoagulation. In all patients, anticoagulation was started≥4 weeks before RFA. Dabigatran was held after the morning dose on the day before the procedure and resumed 4 hours after vascular hemostasis was achieved. Results A transesophageal echocardiogram performed in all patients receiving dabigatran did not demonstrate an intracardiac thrombus. There were no thromboembolic complications in either group. The prevalence of major (4 of 191, 2.1%) and minor (5 of 191, 2.6%) bleeding complications in the dabigatran group were similar to those in the warfarin group (12 of 572, 2.1%; P = 1.0 and 19 of 572, 3.3%; P = .8, respectively). Pericardial tamponade occurred in 2 of 191 (1%) patients in the dabigatran group and in 7 of 572 (1.2%) patients in the warfarin group ( P = 1.0). All patients who had a pericardial tamponade, including 2 in the dabigatran group, had uneventful recovery after perdicardiocentesis. On multivariate analysis, international normalized ratio (odds ratio [OR] 4.0; 95% confidence interval [CI] 1.1–15.0; P = .04), clopidogrel use (OR 4.2; 95% CI 1.5–12.3; P = .01), and CHA 2 DS 2 -VASc score (OR 1.4; 95% CI 1.1–1.8; P = .01) were the independent risk factors of bleeding complications only in the warfarin group. Conclusions When held for approximately 24 hours before the procedure and resumed 4 hours after vascular hemostasis, dabigatran appears to be as safe and effective as uninterrupted warfarin for periprocedural anticoagulation in patients undergoing RFA of AF.

Background: The biochemical pathways underlying glucose-stimulated insulin secretion have not been fully elucidated. Results: Mass spectrometry analysis revealed rapid and substantial metabolic reprogramming evoked by glucose in INS-1 cells. Conclusion: Metabolomics allowed testing and generation of multiple hypotheses regarding glucose effects in insulin-secreting cells. Significance: Insights into the biochemical basis of glucose-stimulated insulin secretion are critical for understanding root causes of type 2 diabetes., Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 to 10 mm glucose and in response to increasing concentrations of glucose in the INS-1 832/13 β-cell line. U-[13C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.

Oxytocin (OXT) and arg-vasopressin (AVP) are nonapeptides with many important functions both peripherally and centrally. Intracerebral microdialysis has helped characterize their importance in regulating complex social and emotional processes. Radioiummunoassay is the most commonly used analytical method used for OXT and AVP measurements in microdialysates. These measurements have several well-known issues including single peptide per assay limit, possible cross-reactivity between structurally related peptides, and laborious sample preparation with radioactive materials. Here we demonstrate the use of capillary LC-MS(3) for measuring OXT and AVP simultaneously in dialysates at a 10 min sampling frequency. Microdialysate samples required no preparation and instrumentation was commercially available. Microdialysis probes made with polyacrylonitrile membranes were suitable for high level recovery of the peptides in vitro and in vivo. Responses were linear from 1 to 100 pM. Matrix effect was assessed by standard addition experiments and by comparing signal intensities of OXT and AVP standards made in aCSF or dialysate. It was determined that the online washing step used on this setup was adequate for removing contaminants which interfere with electrospray ionization efficiency. In vivo, both peptides were stimulated by high K(+) (75 mM) aCSF perfusion in the paraventricular nucleus (PVN). Also, a systemic injection of high Na(+) (2M) caused a rapid and transient increase in PVN OXT while AVP increased only after 1.5h. Our findings suggest that capillary LC-MS(3) is a straightforward method for monitoring OXT and AVP simultaneously from complex samples such as dialysates.

An off-line in vivo neurochemical monitoring approach was developed based on collecting nanoliter microdialysate fractions as an array of “plugs” segmented by immiscible oil in a piece of Teflon tubing. The dialysis probe was integrated with the plug generator in a polydimethlysiloxane microfluidic device that could be mounted on the subject. The microfluidic device also allowed derivatization reagents to be added to the plugs for fluorescence detection of analytes. Using the device, 2 nL fractions corresponding to 1–20 ms sampling times depending upon dialysis flow rate, were collected. Because axial dispersion was prevented between them, each plug acted as a discrete sample collection vial and temporal resolution was not lost by mixing or diffusion during transport. In vitro tests of the system revealed that the temporal resolution of the system was as good as 2 s and was limited by mass transport effects within the dialysis probe. After collection of dialysate fractions, they were pumped into a glass microfluidic chip that automatically analyzed the plugs by capillary electrophoresis with laser-induced fluorescence at 50 s intervals. By using a relatively low flow rate during transfer to the chip, the temporal resolution of the samples could be preserved despite the relatively slow analysis time. The system was used to detect rapid dynamics in neuroactive amino acids evoked by microinjecting the glutamate uptake inhibitor l -trans-pyrrolidine-2,4-dicarboxylic acid (PDC) or K + into the striatum of anesthetized rats. The resulted showed increases in neurotransmitter efflux that reached a peak in 20 s for PDC and 13 s for K + .

Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD(+), which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD(+) metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess nicotinamide mononucleotide 5'-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5'-nucleotidase, and Sdt1, initially classified as a pyrimidine 5'-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD(+). Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD(+) metabolism.

Methods and advances for monitoring neurotransmitters in vivo or for tissue analysis of neurotransmitters over the last five years are reviewed. The review is organized primarily by neurotransmitter type. Transmitter and related compounds may be monitored by either in vivo sampling coupled to analytical methods or implanted sensors. Sampling is primarily performed using microdialysis, but low-flow push-pull perfusion may offer advantages of spatial resolution while minimizing the tissue disruption associated with higher flow rates. Analytical techniques coupled to these sampling methods include liquid chromatography, capillary electrophoresis, enzyme assays, sensors, and mass spectrometry. Methods for the detection of amino acid, monoamine, neuropeptide, acetylcholine, nucleoside, and soluable gas neurotransmitters have been developed and improved upon. Advances in the speed and sensitivity of these methods have enabled improvements in temporal resolution and increased the number of compounds detectable. Similar advances have enabled improved detection at tissue samples, with a substantial emphasis on single cell and other small samples. Sensors provide excellent temporal and spatial resolution for in vivo monitoring. Advances in application to catecholamines, indoleamines, and amino acids have been prominent. Improvements in stability, sensitivity, and selectivity of the sensors have been of paramount interest.

Reversed-phase HPLC was coupled on-line to a rapid, competitive affinity probe capillary electrophoresis (APCE) assay to screen mixtures for compounds that inhibit protein-ligand interactions. The Fyn Src homology 2 (SH2) domain and its phosphopeptide binding partner were used as a model interaction for demonstration of this technique. In the method, mixtures containing possible inhibitors of binding were separated by HPLC at a flow rate of 0.3 mL/min. A small portion of effluent was directed to a fluidic tee where it was mixed on-line with Fyn SH2 domain and a fluorescent phosphopeptide ("affinity probe") known to bind selectively to Fyn SH2 domain. Electropherograms of the reaction mixture were collected on-line at approximately 6s intervals using a flow-gated interface to control injections onto the capillary electrophoresis with laser-induced fluorescence system. The resulting electropherograms contained two peaks, one corresponding to the free affinity probe and the other a complex of the affinity probe and Fyn SH2 domain. Compounds that bound the protein were detected as a decrease in the peak height of the complex and an increase in the peak height of affinity probe with relative standard deviations of5%. The assay was shown to resolve multiple peptidergic inhibitors and selectively detect them within a complex mixture of peptides. Signals were dependent upon both concentration of active peptide and its potency in binding inhibition. Detection limits were in the range of 2-11 microM depending upon the peptide. Common organic solvents used in HPLC were shown to have minimal effect in the on-line measurement up to approximately 60% in the mobile phase.

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 sec) versus a slow (100 sec) 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 seconds. 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 behaviour.

Capillary liquid chromatography coupled with electrospray ionization to a quadropole ion trap mass spectrometer was explored as a method for the analysis of polar anionic compounds in complex metabolome mixtures. A ternary mobile phase gradient, consisting of aqueous acidic, aqueous neutral and organic phases in combination with an aqueous compatible reversed-phase stationary phase allowed metabolites with a wide range of polarities to be resolved and detected. Detection limits in the full scan mode for glycolysis and tricarboxylic acid cycle intermediates were from 0.9 to 36 fmol. Using this system, 111 ± 9 (n = 3) metabolites were detected in Escherichia coli lysate samples. Reducing column I.D. from 50 μm to 25μm increased the number of metabolites detected to 156 ± 17 (n = 3). The improvement in number of metabolites detected was attributed to an increase in separation efficiency, an increase in sensitivity, and a decrease in adduct formation. Implementation of a second separation mode, strong anion exchange, to fractionate the sample prior to capillary RPLC increased the number of metabolites detected to 244 ± 21 (n = 3). This improvement was attributed to the increased peak capacity which decreased co-elution of molecules enabling more sensitive detection by mass spectrometry. This system was also applied to islets of Langerhans where more significant improvements in metabolite detection were observed. In islets, 391 ± 33 small molecules were detected using the two-dimensional separation. The results demonstrate that column miniaturization and use of two-dimensional separations can yield a significant improvement in the coverage of the metabolome.

Over the past decade a tremendous amount of research has been performed using microfluidic analytical devices to detect over 200 different chemical species. Most of this work has involved substantial integration of fluid manipulation components such as separation channels, valves, and filters. This level of integration has enabled complex sample processing on miniscule sample volumes. Such devices have also demonstrated high throughput, sensitivity, and separation performance. Although the miniaturization of fluidics has been highly valuable, these devices typically rely on conventional ancillary equipment such as power supplies, detection systems, and pumps for operation. This auxiliary equipment prevents the full realization of a "lab-on-a-chip" device with complete portability, autonomous operation, and low cost. Integration and/or miniaturization of ancillary components would dramatically increase the capability and impact of microfluidic separations systems. This review describes recent efforts to incorporate auxiliary equipment either as miniaturized plug-in modules or directly fabricated into the microfluidic device.

Capillary liquid chromatography-mass spectrometry (cLC-MS) was coupled on-line to microdialysis sampling to monitor endogenous acetylcholine (ACh) from the rodent brain. In vivo microdialysate sampled at 0.6 microL/min from the striatum of ketamine or chloral hydrate anesthetized rats was loaded onto a sample loop and then injected onto a approximately 5 cm long strong cation exchange (SCX) capillary column. A step gradient was used to separate the analyte from ionization suppressing salts contained in dialysate in 2.4 min. Sampling coupled on-line with cLC-MS allowed for high temporal resolution (data points at 2.4 min intervals), good reproducibility (10-15% relative standard deviation, R.S.D.), and sensitive limits of detection (0.04 nM or 8 amol injected). The method successfully monitored basal and stimulated levels (induced by increased K+ concentrations) of ACh from the anesthetized rat without necessitating perfusion of an acetylcholinesterase (AChE) inhibitor. Absolute and percent basal levels of ACh from rats receiving different anesthetics were also compared.

Capillary electrophoresis (CE) was coupled to negative mode electrospray ionisation-mass spectrometry (MS) for separation and detection of phosphorylated and acidic metabolites in extracts of prokaryotes. Unlike previous CE-MS systems for metabolite analysis, a sheathless interface was used to improve sensitivity. To accomplish this, the separation capillary was modified by creating a porous junction near the outlet where the electrospray voltage and cathodic voltage for CE were applied. The outlet of the capillary was pulled to a 5 microm inner diameter to form an electrospray emitter and had a frit fabricated near the exit to prevent clogging. During analysis pressure was applied at the inlet of the separation column to create sufficient flow towards the detector. Limits of detection for 19 metabolites in full scan mode ranged from 20 nM for ADP ribose to 2.5 microM for alpha-ketoglutarate for 40 nL injections. Extracts of Escherichia coli, strain DH5-alpha, were analyzed using this system. In full scan mode, 118 different metabolites were detected. Tandem mass spectrometry was also employed to attempt identification. Reproducible fragmentation of 19 parent peaks was found and 10 of these produced spectra that were consistent with identification obtained from matching to compounds in the MetaCyc database. These results demonstrate the utility of a sensitive CE-MS system for large scale metabolite detection in biological samples.

Regulation of glucose-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) was investigated by using a novel technique, electroporation from an electrolyte-filled capillary, to deliver energy metabolites to the intracellular compartment of mouse islets. Intracellular application of ATP resulted in a nifedipine-sensitive increase in [Ca2+]i, consistent with a KATP-channel dependent mechanism of Ca2+ influx. [Ca2+]i in islets exposed to 10 mM glucose oscillated with a period of approximately 3 min, often superimposed with faster oscillations. Electroporation of ATP blocked all types of oscillations and elevated [Ca2+]i while delivery of ADP had no effect on oscillations. Intracellular delivery of glucose-6-phosphate or fructose-1,6-bisphosphate tended to transform slow oscillations to fast oscillations. These results demonstrate that modulation of ATP concentrations and glycolytic flux are important in development of slow oscillations.

Glucose induces complex patterns of oscillations in intracellular Ca2+ concentration ([Ca2+]i), metabolism and secretion in islets of Langerhans including "slow" and "fast" pulses with period of 2-5 min and 10-20 s respectively. In an effort to elucidate the origin of slow oscillations, individual mouse islets were exposed to different fuels including glyceraldehyde, pyruvate, methyl pyruvate and alpha-ketoisocaproate (KIC), all of which bypass key steps of glycolytic metabolism, while monitoring [Ca2+]i, oxygen consumption and secretion. Glyceraldehyde gave rise to slow oscillations only when substimulatory glucose was also added to the media. Glucosamine, an inhibitor of glucokinase, blocked these slow oscillations. KIC, pyruvate, and methyl pyruvate did not give rise to slow oscillations alone or with glucose present. The addition of glucose to islets bathed in nutrient-rich cell culture media accelerated metabolism and initiated slow oscillations while glyceraldehyde did not. It is concluded that glucose has a special role in accelerating metabolism and generating slow oscillations in isolated islets of Langerhans from mice. Combined with previous observations of Ca2+ dependency for all oscillations in islets, we propose that interactions between Ca2+ influx and glycolysis are responsible for the slow oscillations. In contrast, fast oscillations can occur independent of glycolytic flux.

An analytical method was developed to monitor amino acids collected by in vivo microdialysis. Microdialysate was continuously derivatized on-line by mixing 6 mM naphthalene-2,3-dicarboxyaldehyde (NDA) and 10 mM potassium cyanide with the dialysate stream in a fused silica capillary to form fluorescent products. Reaction time, determined by the flow rate and volume of reaction capillary, was 3 min. Derivatized amino acids were continuously delivered into a flow-gated interface and periodically injected onto a capillary electrophoresis unit equipped with a laser-induced fluorescence detection based on a commercial microscope. Separation was performed in the micellar electrokinetic chromatography mode using 30 mM sodium dodecyl sulfate in 15 mM phosphate buffer at pH 8.0 as the separation media. An electric field of 1.3 kV/cm was applied across a 10 cm long, 10 microm internal diameter separation capillary. These conditions allowed 17 amino acid derivatives to be resolved in less than 30 s. On-line injections could be performed at 30 s intervals for in vivo samples. Detection limits were from 10 to 30 nM for the amino acids. The method was applied to monitor the acute ethanol-induced amino acid level changes in freely moving rats. The results demonstrate the utility of the method to reveal dynamics of amino acid concentration in vivo.

The development of an efficient method for high-throughput analysis of multiple electropherograms or chromatograms collected in series is presented. The method, encoded in a computer program designated "Cutter", utilizes batch processing for determining chromatographic figures of merit (CFOM) including peak centroid times, heights, areas, signal-to-noise ratios (S/N), variance (sigma2), skew, excess, and plate number (N) across a set of separations collected serially. The software was validated using simulated data with varying S/N, skew, and excess. The accuracy of the analysis was comparable to or improved over commercial software with area calculation relative errors (RE) below 5% for simulated data with S/N = 5. File sets containing 1300 electropherograms were analyzed in 5 min, representing a nearly 200-fold reduction in analysis time from other methods. Incorporated within the program is a novel method for automated peak deconvolution using an Empirically Transformed Gaussian function. Area measurements of deconvoluted peaks were within 3% of the true value of a simulated data set with S/N = 5 and resolution (R(S)) = 1 for equivalent peaks, and within 10% when the ratio of the overlapped peak heights was 10:1.

A glucose oxidase-based glucose microsensor (10 micro m tip diameter) was used to measure the glucose concentration within single islets under static conditions and during step changes in glucose level. The sensors had response times of 4.1+/-0.5s (n=7) and sensitivities of 8.7+/-1.8 pA/mM (n=11). The sensors performed independent of oxygen up to 15 mM glucose as long as the oxygen level was70 mm Hg. Spatially resolved glucose measurements revealed a glucose gradient around and inside single islets. From measurement of the glucose gradient, a glucose consumption rate of 0.48+/-0.14 pmol/nL islet/min (n=6) and an intraislet glucose diffusion coefficient of 3.8 x 10(-7)cm(2)/s were determined. The measurement of the gradient demonstrates that not all cells within an islet in culture are exposed to the same glucose concentration. The sensor was also used to measure the time required for intraislet glucose concentration to reach steady state following a step increase in glucose concentration from 3 to 10 mM at the islet surface. At a depth of 70 micro m inside an islet, glucose reached steady state in 180+/-7s (n=7) for islets with a diameter of 180-220 micro m (smaller islets reach steady-state faster). In the presence of 10 mM mannoheptulose, an inhibitor of glucokinase, the equilibration time was reduced to 122+/-11s (n=6), indicating that glucose utilization by glycolysis limited the time required for glucose to diffuse into the islets. The long times to reach steady state and presence of glucose gradients are important in interpreting data from experiments involving islets in culture.

A method for the separation and quantitative determination of neuroactive amino acids (aspartate, glutamate, citrulline, arginine, glycine, taurine, gamma-aminobutyric acid) and neuroactive amines (noradrenaline, dopamine and serotonin) in a single chromatographic analysis is presented. The method is based on pre-column derivatization with o-phthalaldehyde and tert.-butyl thiol, on-column preconcentration and separation using 50 microns I.D. packed capillary columns, and detection by amperometry. Mass limits of detection are 80-900 amol for all neurotransmitters with RSDs of 0.71 and 4.6% or better for retention time and peak area, respectively. The method was demonstrated by application to the determination of neurotransmitters in microdialysis samples collected from striatum of live rats and tissue samples extracted from butterfly brains.

Immunoreactions in combination with separations by capillary electrophoresis (CE) are increasingly being used to quantitate specific analytes in biological fluids. Both competitive and non-competitive approaches have been used for the purpose and, in selected cases, now compare favorably with conventional quantitative immunoassays with respect to concentration limits of detection. CE is also a useful method to evaluate antigen–antibody binding on-line and offers unique possibilities for binding constant estimates, also for weakly binding antibodies and antibody fragments. In this review we cover recent developments in the use of antigen–antibody interactions in conjunction with CE and conclude that continued development of miniaturization, on-line preconcentration and more sensitive detection schemes will contribute to the further dissemination of CE-based immunoassays building on already established affinity CE approaches.

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.

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.

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.

Capillary electrophoresis is unique among liquid-phase separations in its utility for fast separations. Development of technology such as optical-gating, flow-gating, and microfabrication has allowed separations on the millisecond time scale to be developed. The fast separation times place great demands on the detector systems, frequently requiring detection limits below 1 amol to be practical. The development of such fast separations has opened many new applications not previously feasible for separations-based methods. This has included real time chemical monitoring, detection of short-lived species such as protein conformers or non-covalent complexes, and rapid multi-dimensional separations. Other applications currently being developed include high-throughput assays for clinical laboratories or screening combinatorial libraries. This review covers recent developments in the instrumentation for fast CE and some of the applications.

A novel oxygen microsensor was used to measure oxygen levels in single mouse islets as a function of glucose concentration. Oxygen consumption of individual islets was 5.99 +/- 1.17, 9.21 +/- 2.15, and 12.22 +/- 2.16 pmol/min at 3, 10, and 20 mM glucose, respectively (mean +/- SEM, n = 10). Consumption of oxygen was islet-size dependent as larger islets consumed more oxygen than smaller islets but smaller islets consumed more oxygen per unit volume than larger islets. Elevating glucose levels from 3 to 10 mM induced pronounced fast oscillations in oxygen level (period of 12.1 +/- 1.7 s, n = 6) superimposed on top of large slow oscillations (period of 3.3 +/- 0.6 min, n = 6). The fast oscillations could be completely abolished by treatment with the L-type Ca2+-channel blocker nifedipine (40 microM) with a lesser effect on slow oscillations. Slow oscillations were almost completely dependent upon extracellular Ca2+. The oxygen patterns closely mimic those that have previously been reported for intracellular Ca2+ levels and are suggestive of an important role for Ca2+ in amplifying metabolic oscillations.

Laser-induced fluorescence (LIF) has been used extensively in capillary separations due to its high sensitivity and selectivity. This article highlights the history and recent advances and applications of LIF detection in capillary electrophoresis and capillary chromatography.

The effects of extracellular Zn2+ and pH and intravesicular pH on insulin and 5-hydroxytryptamine (5-HT) secretion from pancreatic beta cells were investigated. Insulin and 5-HT secretion from single cells was detected by amperometry as a series of current spikes corresponding to detection of multimolecular packets secreted by exocytosis. Spike width was used as a measure of the kinetics of clearance from the cell and the area of spikes as a measure of amount released. Changes in extracellular pH from 6.9 to 7.9 caused insulin spikes to become narrower with no change in area, whereas the same treatments had no effect on 5-HT secretion. Treatment of cells with Bafilomycin A1 or N-ethylmaleimide, both of which are expected to increase intravesicular pH by inhibiting V-type H+-ATPase, had no effect on 5-HT secretion but caused insulin spikes to become more narrow. These results indicate that exposure to high pH, whether intravesicular or extracellular, accelerates release of insulin during exocytosis without affecting the amount of insulin released. Increasing extracellular Zn2+ concentration from 0 to 25 microM increased the width and decreased the area of insulin spikes without affecting 5-HT secretion. Zn2+ effects were likely exerted through a common-ion effect on Zn2+-insulin dissociation. It was concluded that intravesicular storage conditions and extracellular ions can affect free insulin concentration in the vicinity of beta cells during secretion.

Flow-through recycling electrochemical detectors with dual Au electrodes were fabricated using micromachining techniques. Detector cells contained two microband electrodes that were 50 μm wide by 3–5 mm long separated by a 5 or 10 μm gap in a parallel-opposed configuration. One electrode was defined in the bottom of a channel which was etched into the surface of a Si wafer while the other electrode was defined on a glass substrate. The depth of the channel defined the gap distance between the electrodes. The Si and glass pieces were anodically bonded together to create a flow cell. The stability and reproducibility of single and dual electrode cells were characterized with cyclic voltammetry and amperometry during flow injection analysis (FIA). Single electrode detectors had detection limits of 50 nM with root mean square noise in the 3–10 pA range. The dual electrode cells showed enhanced sensitivity over single electrode cells through detection of analyte molecules multiple times as they were transported through the cell. Signal enhancements up to 60-fold were obtained with dual electrode cells during FIA at a flow rate of 50 nl/min. Simulations predict that signal-to-noise ratio enhancements > 100-fold may be possible with appropriate designs.

A ruthenium-oxide-type catalytic film (RuOx) was produced on carbon fiber microelectrodes by cycling the electrode potential between 0.65 and −0.85V vs. SSCE at 100 V s−1 in an air-equilibrated acidic solution of RuCl3. The film catalyzes oxidation of insulin in a saline buffer at pH 7.4. The minimum number of electrons transferred during the insulin oxidation at 0.65 V is 6.7. The analytical performance of the modified electrode as an amperometric detector for insulin was characterized using flow injection analysis. Linear least squares calibration curves over the range 0.10 to 1.0 μM (five points) had slopes of 72 ± 2 pA μM−1 and correlation coefficients of 0.999 or greater. The detection limit, calculated as the concentration that would yield a signal equal to three times the root mean square noise, was 23 nM and response time (t90%) was 40 ms or less. The electrode response to 0.2 μM insulin was stable for 3 days. The modified electrode was used for amperometric detection of exocytosis from individual pancreatic s-cells.

A system which couples microdialysis with capillary zone electrophoresis (CZE) on-line is used to monitor ascorbate and lactate in the caudate nucleus of rat brain. On-line interface of microdialysis probe and electrophoresis capillary, along with the high mass sensitivity of CZE, allows the probe to be operated at flow rates as low as 40 nl/min. Under these conditions, the relative recovery is nearly 100% and quantitative monitoring is possible. The microscale system also facilitates calibration by the low flow rate method. In spite of the low flow rate, temporal resolution in the 45-125 s range is possible for these compounds. The system is demonstrated by observing changes in ascorbate due to infusions of elevated K+ through the dialysis probe and systemic injections of amphetamine and an anesthetic (ketamine/xylazine/acepromazine mixture). Lactate is monitored in response to elevated K+ infusions.

A flow-gated, on-line interface between a microdialysis sampling probe and capillary zone electrophoresis with UV absorption detection was characterized and applied. Electrophoresis conditions were chosen so that ascorbic acid migrated in 42–100 s with 65000–120000 theoretical plates. These results were obtained using a 25 μm inner diameter capillary with an inlet to detector length of 15 cm and electric field strength of 400–600 V cm −1 . Theoretical plates, migration times, and peak areas for ascorbic acid injected on-line from the interface were the same as those obtained for off-line injections. The system allowed step changes in ascorbic acid concentration to be followed with 85 s time resolution when the dialysis flow-rate was 79 nl min −1 . The time resolution was improved to 65 s when the dialysis flow-rate was 155 ni min −1 . The relative standard deviation for peak heights was 3.8% and a linear response over the physiologically relevant range for ascorbic acid was observed. At 79 nl min −1 the relative recovery of the probe was 98%. The high relative recoveries improved detection limits, simplified quantification, and resulted in decreased disturbance to the system being studied when compared to the more conventional dialysis flow-rates of 0.5-1 μl min −1 . As a demonstration of the system, ascorbic acid in the caudate nucleus of rat brain was detected and monitored in response to systemic amphetamine injections and anaesthetic overdoses. This system is the first to demonstrate high relative recoveries and high time resolution simultaneously with microdialysis sampling.

Microelectrodes have found application for monitoring the chemical environment of single cells. To extend the utility of electrochemical methods applied to single-cell analysis, it has been necessary to use chemically modified microelectrodes which allow detection of substances with slow redox kinetics. In this article, we discuss the use of chemically modified microelectrodes for detection of insulin secretion, nitric oxide secretion, and glucose metabolism inside single cells.