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1.  Chemical Gradients within Brain Extracellular Space Measured using Low Flow Push–Pull Perfusion Sampling in Vivo 
ACS Chemical Neuroscience  2012;4(2):321-329.
Although populations of neurons are known to vary on the micrometer scale, little is known about whether basal concentrations of neurotransmitters also vary on this scale. We used low-flow push–pull perfusion to test if such chemical gradients exist between several small brain nuclei. A miniaturized polyimide-encased push–pull probe was developed and used to measure basal neurotransmitter spatial gradients within brain of live animals with 0.004 mm3 resolution. We simultaneously measured dopamine (DA), norepinephrine, serotonin (5-HT), glutamate, γ-aminobutyric acid (GABA), aspartate (Asp), glycine (Gly), acetylcholine (ACh), and several neurotransmitter metabolites. Significant differences in basal concentrations between midbrain regions as little as 200 μm apart were observed. For example, dopamine in the ventral tegmental area (VTA) was 4.8 ± 1.5 nM but in the red nucleus was 0.5 ± 0.2 nM. Regions of high glutamate concentration and variability were found within the VTA of some individuals, suggesting hot spots of glutamatergic activity. Measurements were also made within the nucleus accumbens core and shell. Differences were not observed in dopamine and 5-HT in the core and shell; but their metabolites homovanillic acid (460 ± 60 nM and 130 ± 60 nM respectively) and 5-hydroxyindoleacetic acid (720 ± 200 nM and 220 ± 50 nM respectively) did differ significantly, suggesting differences in dopamine and 5-HT activity in these brain regions. Maintenance of these gradients depends upon a variety of mechanisms. Such gradients likely underlie highly localized effects of drugs and control of behavior that have been found using other techniques.
PMCID: PMC3582294  PMID: 23421683
Dopamine; glutamate; push−pull perfusion; microdialysis; spatial resolution; in vivo
2.  CNS penetration of the opioid glycopeptide MMP-2200: A microdialysis study 
Neuroscience letters  2012;531(2):99-103.
Endogenous opioid peptides enkephalin and dynorphin are major co-transmitters of striatofugal pathways of the basal ganglia. They are involved in the genesis of levodopa-induced dyskinesia and in the modulation of direct and indirect striatal output pathways that are disrupted in Parkinson’s disease. One pharmacologic approach is to develop synthetic glycopeptides closely resembling endogenous peptides to restore their normal functions. Glycosylation promotes penetration of the blood-brain barrier. We investigated CNS penetration of the opioid glycopeptide MMP-2200, a mixed δ/μ-agonist based on leu-enkephalin, as measured by in vivo microdialysis and subsequent mass spectrometric analysis in awake, freely moving rats. The glycopeptide (10 mg/kg) reaches the dorsolateral striatum (DLS) rapidly after systemic (i.p.) administration and is stably detectable for the duration of the experiment (80 min). The detected level at the end of the experiment (around 250 pM) is about 10-fold higher than the level of the endogenous leu-enkephalin, measured simultaneously. This is one of the first studies to directly prove that glycosylation of an endogenous opioid peptide leads to excellent blood-brain barrier penetration after systemic injection, and explains robust behavioral effects seen in previous studies by measuring how much glycopeptide reaches the target structure, in this case the DLS.
PMCID: PMC3539793  PMID: 23127847
striatum; leu-enkephalin; glycopeptide; blood-brain barrier penetration; mass spectrometry
3.  Enkephalin surges in dorsal neostriatum as a signal to eat 
Current biology : CB  2012;22(20):1918-1924.
Compulsive over-consumption of rewards characterizes disorders ranging from binge eating to drug addiction. Here, we provide evidence that enkephalin surges in an anteromedial quadrant of dorsal neostriatum contribute to generating intense consumption of palatable food. In ventral striatum, mu opioid circuitry contributes an important component of motivation to consume rewards [1–4]. In dorsal neostriatum, mu opioid receptors are concentrated within striosomes that receive inputs from limbic regions of prefrontal cortex [5–13]. We employed advanced opioid microdialysis techniques that allow detection of extracellular enkephalin levels. Endogenous >150% enkephalin surges in anterior dorsomedial neostriatum were triggered as rats began to consume palatable chocolates. By contrast, dynorphin levels remained unchanged. Further, a causal role for mu opioid stimulation in over-consumption was demonstrated by observations that microinjection in the same anterior dorsomedial quadrant of a mu receptor agonist (DAMGO) generated intense >250% increases in intake of palatable sweet food (without altering hedonic impact of sweet tastes). Mapping by “Fos plume” methods confirmed the hyperphagic effect to be anatomically localized to the anterior medial quadrant of the dorsal neostriatum, whereas other quadrants were relatively ineffective. These findings reveal that opioid signals in anteromedial dorsal neostriatum are able to code and cause motivation to consume sensory rewards.
PMCID: PMC3482294  PMID: 23000149
4.  Simultaneous oxytocin and arg-vasopressin measurements in microdialysates using capillary liquid chromatography-mass spectrometry 
Journal of Neuroscience Methods  2012;209(1):127-133.
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-MS3 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 – 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 1.5 h. Our findings suggest that Capillary LC-MS3 is a straightforward method for monitoring OXT and AVP simultaneously from complex samples such as dialysates.
PMCID: PMC3402657  PMID: 22710285
oxytocin; vasopressin; microdialysis; mass spectrometry; paraventricular nucleus; hypothalamus
5.  A Mass Spectrometry “Sensor” for in Vivo Acetylcholine Monitoring 
Analytical Chemistry  2012;84(11):4659-4664.
Developing sensors for in vivo chemical monitoring is a daunting challenge. An alternative approach is to couple sampling methods with online analytical techniques; however, such approaches are generally hampered by lower temporal resolution and slow analysis. In this work, microdialysis sampling was coupled with segmented flow electrospray ionization mass spectrometry (ESI-MS) to perform in vivo chemical monitoring. Use of segmented flow to prevent Taylor dispersion of collected zones and rapid analysis with direct ESI-MS allowed 5 s temporal resolution to be achieved. The MS “sensor” was applied to monitoring acetylcholine in the brain of live rats. The detection limit of 5 nM was sufficient to monitor basal acetylcholine as well as dynamic changes elicited by microinjection of neostigmine, an inhibitor of acetycholinesterase that evoked rapid increases in acetycholine, and tetrodotoxin, a blocker of Na+ channels, that lowered the acetylcholine concentration. The versatility of the sensor was demonstrated by simultaneously monitoring metabolites and infused drugs.
PMCID: PMC3389145  PMID: 22616788
6.  Analysis of Fatty Acid Composition in Insulin Secreting Cells by Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry 
A comprehensive two-dimensional gas chromatography (GC×GC) time-of-flight mass spectrometry method was developed for determination of fatty acids (irrespective of origin i.e., both free fatty acids and fatty acids bound in sources such as triglycerides) in cultured mammalian cells. The method was applied to INS-1 cells, an insulin-secreting cell line commonly used as a model in diabetes studies. In the method, lipids were extracted and transformed to fatty acid methyl esters for analysis. GC×GC analysis revealed the presence of 30 identifiable fatty acids in the extract. This result doubles the number of fatty acids previously identified in these cells. The method yielded linear calibrations and an average relative standard deviation of 8.4 % for replicate injections of samples and 12.4 % for replicate analysis of different samples. The method was used to demonstrate changes in fatty acid content as a function of glucose concentration on the cells. These results demonstrate the utility of this method for analysis of fatty acids in mammalian cell cultures.
PMCID: PMC3322247  PMID: 22456534
lipids; GCxGC; insulin
Analytical and Bioanalytical Chemistry  2012;402(9):2797-2803.
A rapid microfluidic based capillary electrophoresis immunoassay (CEIA) was developed for on-line monitoring of glucagon secretion from pancreatic islets of Langerhans. In the device, a cell chamber containing living islets was perfused with buffers containing either high or low glucose concentration. Perfusate was continuously sampled by electroosmosis through a separate channel on the chip. The perfusate was mixed on-line with fluorescein isothiocyanate-labeled glucagon (FITC-glucagon) and monoclonal anti-glucagon antibody. To minimize sample dilution, the on-chip mixing ratio of sampled perfusate to reagents was maximized by allowing reagents to only be added by diffusion. Every 6 s the reaction mixture was injected onto a 1.5 cm separation channel where free FITC-glucagon and the FITC-glucagon-antibody complex were separated under an electric field of 700 V cm−1. The immunoassay had a detection limit of 1 nM. Groups of islets were quantitatively monitored for changes in glucagon secretion as the glucose concentration was decreased from 15 to 1 mM in the perfusate revealing a pulse of glucagon secretion during a step change. The highly automated system should be enable studies of the regulation of glucagon and its potential role in diabetes and obesity. The method also further demonstrates the potential of rapid CEIA on microfluidic systems for monitoring cellular function.
PMCID: PMC3324330  PMID: 22286080
electrophoresis; immunoassay; cells on chips; microfluidics; glucagon
8.  Factors Associated With Sexually Transmitted Infections in Men and Women 
Sexually transmitted infections (STIs) remains a serious healthcare problem costing approximately 13 billion dollars annually to treat. Men and women who contract STIs have a higher risk for reinfection and for developing human immunodeficiency virus (HIV). Determining the risk factors associated with STIs in a community would be helpful in designing culturally appropriate tailored interventions to reduce spread of STIs.
The purpose of this retrospective chart review was to determine the frequency and type of STIs, as well as to determine the predictor variables associated with STIs among those seeking treatment at a local inner city health unit.
A total of 237 medical records were reviewed from a STI clinic. The sample comprised 119 men and 118 women, of whom 70.9% were African American. The mean age was 27, and 38% had a prior STI. Men used significantly more condoms (χ2 = 24.28, p = 0.000), had more sexual partners (χ2 =18.36, p = 0.003), and had more prior infections of gonorrhea (χ2 = 10.04, p =0.002) than women. Women had significantly more prior infections of Chlamydia (χ2 = 11.74, p = 0.001). Using no type of birth control measures (pills, diaphragm, implants) was a significant predictor of number of sexual partners (t = 2.441, p < 0.015), but negatively associated with condom use (t = −12.290, p < 0.000).
Over one-third had a prior STI, indicating that individuals do not perceive themselves to be at risk for another STI, and choose not to use condoms. Reasons why individuals continue to put themselves at risk need to be explored in gender specific focus groups so that tailored sexual risk reduction programs can be designed to meet the needs of different communities.
PMCID: PMC3582223  PMID: 19662560
9.  Metabolome Response to Glucose in the β-Cell Line INS-1 832/13* 
The Journal of Biological Chemistry  2013;288(15):10923-10935.
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.
PMCID: PMC3624472  PMID: 23426361
Beta Cell; Insulin Secretion; Intermediary Metabolism; Metabolism; Metabolomics
10.  In Vivo Neurochemical Monitoring using Benzoyl Chloride Derivatization and Liquid Chromatography – Mass Spectrometry 
Analytical Chemistry  2011;84(1):412-419.
In vivo neurochemical monitoring using microdialysis sampling is important in neuroscience because it allows correlation of neurotransmission with behavior, disease state, and drug concentrations in the intact brain. A significant limitation of current practice is that different assays are utilized for measuring each class of neurotransmitter. We present a high performance liquid chromatography (HPLC) - tandem mass spectrometry method that utilizes benzoyl chloride for determination of the most common low molecular weight neurotransmitters and metabolites. In this method, 17 analytes were separated in 8 minutes. The limit of detection was 0.03–0.2 nM for monoamine neurotransmitters, 0.05–11 nM for monoamine metabolites, 2–250 nM for amino acids, 0.5 nM for acetylcholine, 2 nM for histamine, and 25 nM for adenosine at sample volume of 5 µL. Relative standard deviation for repeated analysis at concentrations expected in vivo averaged 7% (n = 3). Commercially available 13C benzoyl chloride was used to generate isotope-labeled internal standards for improved quantification. To demonstrate utility of the method for study of small brain regions, the GABAA receptor antagonist bicuculline (50 µM) was infused into rat ventral tegmental area while recording neurotransmitter concentration locally and in nucleus accumbens, revealing complex GABAergic control over mesolimbic processes. To demonstrate high temporal resolution monitoring, samples were collected every 60 s while neostigmine, an acetylcholine esterase inhibitor, was infused into the medial prefrontal cortex. This experiment revealed selective positive control of acetylcholine over cortical glutamate.
PMCID: PMC3259198  PMID: 22118158
Neurotransmitter; Microdialysis; Benzoylation; Liquid Chromatography; Mass Spectrometry
11.  Microdialysis and Mass Spectrometric Monitoring of Dopamine and Enkephalins in the Globus Pallidus Reveal Reciprocal Interactions that Regulate Movement 
Journal of neurochemistry  2011;118(1):24-33.
Pallidal dopamine, GABA and the endogenous opioid peptides enkephalins have independently been shown to be important controllers of sensorimotor processes. Using in vivo microdialysis coupled to liquid chromatography-mass spectrometry (LC-MS) and a behavioral assay, we explored the interaction between these three neurotransmitters in the rat globus pallidus. Amphetamine (3 mg/kg i.p.) evoked an increase in dopamine, GABA and methionine/leucine enkephalin. Local perfusion of the dopamine D1 receptor antagonist SCH 23390 (100 μM) fully prevented amphetamine stimulated enkephalin and GABA release in the globus pallidus and greatly suppressed hyperlocomotion. In contrast, the dopamine D2 receptor antagonist raclopride (100 μM) had only minimal effects suggesting a greater role for pallidal D1 over D2 receptors in the regulation of movement. Under basal conditions, opioid receptor blockade by naloxone perfusion (10 μM) in the globus pallidus stimulated GABA and inhibited dopamine release. Amphetamine-stimulated dopamine release and locomotor activation were attenuated by naloxone perfusion with no effect on GABA. These findings demonstrate a functional relationship between pallidal dopamine, GABA and enkephalin systems in the control of locomotor behavior under basal and stimulated conditions. Moreover, these findings demonstrate the usefulness of LC-MS as an analytical tool when coupled to in vivo microdialysis.
PMCID: PMC3112281  PMID: 21534957
Globus pallidus; dopamine; enkephalins; mass spectrometry; microdialysis; amphetamine
12.  Push-pull perfusion sampling with segmented flow for high temporal and spatial resolution in vivo chemical monitoring 
Analytical chemistry  2011;83(13):5207-5213.
Low-flow push-pull perfusion is a sampling method that yields better spatial resolution than competitive methods like microdialysis. Because of the low flow rates used (50 nL/min) it is challenging to use this technique at high temporal resolution which requires methods of collecting, manipulating, and analyzing nanoliter samples. High temporal resolution also requires control of Taylor dispersion during sampling. To meet these challenges, push-pull perfusion was coupled with segmented flow to achieve in vivo sampling at 7 s temporal resolution at 50 nL/min flow rates. By further miniaturizing the probe inlet, sampling with 200 ms resolution at 30 nL/min (pull only) was demonstrated in vitro. Using this method, L-glutamate was monitored in the striatum of anesthetized rats. Up to 500 samples of 6 nL each were collected at 7 s intervals, segmented by an immiscible oil and stored in a capillary tube. The samples were assayed offline for L-glutamate at a rate of 15 samples/min by pumping them into a reagent addition tee fabricated from Teflon where reagents were added for a fluorescent enzyme assay. Fluorescence of the resulting plugs was monitored downstream. Microinjection of 70 mM potassium in physiological buffered saline evoked L-glutamate concentration transients that had an average maxima of 4.5 ± 1.1 μM (n = 6 animals, 3–4 injections each) and rise times of 22 ± 2 s. These results demonstrate that low-flow push-pull perfusion with segmented flow can be used for high temporal resolution chemical monitoring and in complex biological environments.
PMCID: PMC3128237  PMID: 21604670
13.  Collection, storage, and electrophoretic analysis of nanoliter microdialysis samples collected from awake animals in vivo 
Analytical and bioanalytical chemistry  2011;400(7):2013-2023.
Microdialysis sampling is an important tool for chemical monitoring in living systems. Temporal resolution is an important figure of merit that is determined by sampling frequency, assay sensitivity, and dispersion of chemical zones during transport from sampling device to fraction collector or analytical system. Temporal resolution has recently been improved by segmenting flow into plugs, so that nanoliter fractions are collected at intervals of 0.1–2 s, thus eliminating temporal distortion associated with dispersion in continuous flow. Such systems, however, have yet to be used with behaving subjects. Furthermore, long-term storage of nanoliter samples created by segmented flow has not been reported. In this work, we have addressed these challenges. A microdialysis probe was integrated to a plug generator that could be stably mounted onto behaving animals. Long-term storage of dialysate plugs was achieved by collecting plugs into high-purity perfluoroalkoxy tubes, placing the tube into hexane and then freezing at −80°C. Slow warming with even temperatures prevented plug coalescence during sample thawing. As a demonstration of the system, plugs were collected from the striatum of behaving rats using a 0.5-mm-long microdialysis probe. Resulting plugs were analyzed 1–4 days later by chip-based electrophoresis. To improve throughput of plug analysis over previous work, the speed of electrophoretic separation was increased by using forced air cooling and 1-butyl-2,3-dimethylimidazolium tetrafluoroborate as a separation buffer additive, allowing resolution of six neuroactive amino acids in 30 s. Concentration changes induced by K+ microinjections were monitored with 10 s temporal resolution. The improvements reported in this work make it possible to apply segmented flow microdialysis to the study of behaving animals and enable experiments where the analytical system cannot be placed close to the animal.
PMCID: PMC3107505  PMID: 21465093
Behaving; Electrophoresis; Microdialysis; Offline analysis; Plug storage; Segmented flow; Spatial resolution; Temporal resolution
14.  Fatigue, Sleep, Pain, Mood and Performance Status in Patients with Multiple Myeloma 
Cancer nursing  2011;34(3):219-227.
Cancer-related fatigue and insomnia are common distressing symptoms and may affect mood and performance status.
to describe fatigue, sleep, pain, mood and performance status and the relationships among these variables in 187 patients newly diagnosed with multiple myeloma (MM) and conduct an analysis using the correlates of fatigue.
Data were from baseline measures from the study, using the Profile of Mood States (POMS) and the Functional Assessment of Cancer Therapy - Fatigue to assess fatigue, the Actigraph to measure sleep, the Wong/Baker Faces Pain Rating Scale to assess pain, the POMS to assess mood, and the 6-minute walk test along with a back/leg/chest dynamometer to test muscle strength to assess performance status. Data analysis consisted of descriptive statistics, Pearson and Spearman rho correlations and multiple regression using fatigue as the dependent variable. All p values were two-sided, and those with < .05 considered significant.
Patients newly diagnosed with MM presented with fatigue, pain, sleep and mood disturbances, and diminished functional performance. The regression model, which included all of these variables along with age, gender and stage of disease, was statistically significant with a large measure of effect. Mood was a significant individual contributor to the model.
Among patients with MM, fatigue, pain, sleep, mood and functional performance are interrelated.
Implications for Practice
Interventions are needed to decrease fatigue and pain and to improve sleep, mood and functional performance.
PMCID: PMC3086027  PMID: 21522061
15.  Reducing Time and Increasing Sensitivity in Sample Preparation for Adherent Mammalian Cell Metabolomics 
Analytical chemistry  2011;83(9):3406-3414.
A simple, fast, and reproducible sample preparation procedure was developed for relative quantification of metabolites in adherent mammalian cells using the clonal β-cell line INS-1 as a model sample. The method was developed by evaluating the effect of different sample preparation procedures on high performance liquid chromatography- mass spectrometry quantification of 27 metabolites involved in glycolysis and the tricarboxylic acid cycle on a directed basis as well as for all detectable chromatographic features on an undirected basis. We demonstrate that a rapid water rinse step prior to quenching of metabolism reduces components that suppress electrospray ionization thereby increasing signal for 26 of 27 targeted metabolites and increasing total number of detected features from 237 to 452 with no detectable change of metabolite content. A novel quenching technique is employed which involves addition of liquid nitrogen directly to the culture dish and allows for samples to be stored at −80 °C for at least 7 d before extraction. Separation of quenching and extraction steps provides the benefit of increased experimental convenience and sample stability while maintaining metabolite content similar to techniques that employ simultaneous quenching and extraction with cold organic solvent. The extraction solvent 9:1 methanol: chloroform was found to provide superior performance over acetonitrile, ethanol, and methanol with respect to metabolite recovery and extract stability. Maximal recovery was achieved using a single rapid (~1 min) extraction step. The utility of this rapid preparation method (~5 min) was demonstrated through precise metabolite measurements (11% average relative standard deviation without internal standards) associated with step changes in glucose concentration that evoke insulin secretion in the clonal β-cell line INS-1.
PMCID: PMC3094105  PMID: 21456517
metabolomics; liquid chromatography-mass spectrometry; adherent mammalian cell sample preparation; INS-1; β-cells; HILIC; solvent; extraction time
16.  Western Blotting using Capillary Electrophoresis 
Analytical chemistry  2011;83(4):1350-1355.
A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ~1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot.
PMCID: PMC3075063  PMID: 21265514
17.  Leptin promotes dopamine transporter and tyrosine hydroxylase activity in the nucleus accumbens of Sprague-Dawley rats 
Journal of neurochemistry  2010;114(3):666-674.
Adipocytes produce the hormone, leptin, in proportion to fat mass to signal the status of body energy stores to the central nervous system, thereby modulating food intake and energy homeostasis. In addition to controlling satiety, leptin suppresses the reward value of food, which is controlled by the mesolimbic dopamine (DA) system. Previous results from leptin-deficient ob/ob animals suggest that chronic leptin deficiency decreases DA content in the mesolimbic DA system, thereby decreasing the response to amphetamine (AMPH). The extent to which these alterations in the mesolimbic DA system of ob/ob animals may mirror the leptin response of normal animals has remained unclear, however. We therefore examined the potential short-term modulation of the mesolimbic DA system by leptin in normal animals. We show that 4 h of systemic leptin treatment enhances AMPH-stimulated DA efflux in the nucleus accumbens (NAc) of Sprague-Dawley rats. While acute leptin treatment increased NAc tyrosine hydroxylase (TH) activity, total TH and DA content were unchanged at this early time point. Leptin also increased NAc DA transporter (DAT) activity in the absence of changes in cell surface or total DAT. Thus, leptin modulates the mesolimbic DA system via multiple acute mechanisms, and increases AMPH-mediated DA efflux in normal animals.
PMCID: PMC2910163  PMID: 20412389
leptin; dopamine; amphetamine; tyrosine hydroxylase; dopamine transporter
Analytical and bioanalytical chemistry  2010;397(7):2939-2947.
A three-layer microfluidic device was developed that combined perfusion of cultured cells with on-line chemical analysis for near real-time monitoring of cellular secretions. Two layers were reversibly sealed to form a cell chamber that could allow cells grown on coverslips to be loaded directly into the chip. The outlet of the chamber was in fluidic contact with a third layer that was permanently bonded. Perfusate from the cell chamber flowed into this third layer where a fluorescence enzyme assay for non-esterified fatty acid (NEFA) was performed on-line. The device was used to monitor efflux of NEFAs from cultured adipocytes with 83 s temporal resolution. Perfusion of murine 3T3-L1 cultured adipocytes resulted in an average basal concentration of 24.2 ± 2.4 μM NEFA (SEM, n = 6) detected in the effluent corresponding to 3.31 × 10−5 nmol cell−1 min−1. Upon pharmacological treatment with a β-adrenergic agonist to stimulate lipolysis, a 6.9 ± 0.7-fold (SEM, n = 6) sustained increase in NEFA secretion was observed. This multilayer device was capable of monitoring NEFA from ~6 250 adipocytes This multilayer device provides a versatile platform that could be adapted for use with other cell types to study corresponding cellular secretions.
PMCID: PMC3125973  PMID: 20549489
Adipocytes; microfluidics; enzyme assay; integration
19.  Referral patterns, clinical examination and the two-week-rule for breast cancer: a cohort study 
The Ulster Medical Journal  2011;80(2):68-71.
Current NHS guidelines require patients with suspected breast cancer to be seen urgently at a specialist breast clinic. The aim of this study was to assess referral patterns and clinical findings of patients referred to a specialist breast clinic.
Materials and Methods
A prospective database was maintained for consecutive patients referred. Symptoms and clinical findings in primary and secondary care were recorded. Correlation with final diagnoses was made. Tertiary referral patients were excluded.
1098 patients attended a specialist breast clinic over six months. 588 (54%) were referred as urgent, 285 (26%) routinely and 225 (20%) were unspecified. 492 (45%) patients were referred with the incorrect referral priority. 42 patients were unexamined in primary care. Examination findings in primary and secondary care correlated in only 487 (46%) patients. Examination in primary care when compared with secondary care was highly sensitive for detecting breast lumps, but specificity was low. 86 patients (8%) were diagnosed with breast cancer, 72 (84%) were referred urgently, 6 (7%) routinely and 8 (9%) as unspecified priority. Regardless of the clinical expertise of the referrer, sensitivity and specificity of the two-week guidelines for cancer are low.
Examination findings in primary and secondary care correlate in only 46% of referrals. Additionally, 55% of referrals were of the correct priority. The two-week rule guidelines have poor sensitivity and specificity for cancer. The safest and fairest policy would be to abandon the concept of urgent referral criteria and see all patients in a timely fashion. Alternatively, simplifying the referral criteria would improve sensitivity and specificity for cancer without leading to increased waiting times.
PMCID: PMC3229848  PMID: 22347745
Breast neoplasms; referral and consultation; guidelines
20.  Collection of nanoliter microdiaysate fractions in plugs for off-line in vivo chemical monitoring with up to 2 s temporal resolution 
Journal of neuroscience methods  2010;190(1):39-48.
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+.
PMCID: PMC2885530  PMID: 20447417
Microdialysis; Segmented flow; Off-line analysis; Temporal resolution; Electrophoresis; Amino acids
21.  Fraction Collection from Capillary Liquid Chromatography and Off-line Electrospray Ionization Mass Spectrometry using Oil Segmented Flow 
Analytical chemistry  2010;82(12):5260-5267.
Off-line analysis and characterization of samples separated by capillary liquid chromatography (LC) has been problematic using conventional approaches to fraction collection. We demonstrate collection of nanoliter fractions by forming plugs of effluent from a 75 μm inner diameter LC column segmented by an immiscible oil such as perfluorodecalin. The plugs are stored in tubing that can then be used to manipulate the samples. Off-line electrospray ionization mass spectrometry (ESI-MS) was used to characterize the samples. ESI-MS was performed by directly pumping the segmented plugs into a nanospray emitter tip. Critical parameters including the choice of oils, ESI voltage, and flow rates that allows successful direct infusion analysis were investigated. Best signals were obtained under conditions in which the oil did not form an electrospray but was siphoned away from the tip. Off-line analysis showed preservation of the chromatogram with no loss of resolution. The method was demonstrated to allow changes in flow rate during the analysis. Specifically, decreases in flow rate were used to allow extended MS analysis time on selected fractions, similar to “peak parking”.
PMCID: PMC2894538  PMID: 20491430
22.  Microfluidic Chip for High Efficiency Electrophoretic Analysis of Segmented Flow from a Microdialysis Probe and in Vivo Chemical Monitoring 
Analytical chemistry  2009;81(21):9072-9078.
An effective method for in vivo chemical monitoring is to couple sampling probes, such as microdialysis, to on-line analytical methods. A limitation of this approach is that in vivo chemical dynamics may be distorted by flow and diffusion broadening during transfer from sampling probe to analytical system. Converting a homogenous sample stream to segmented flow can prevent such broadening. We have developed a system for coupling segmented microdialysis flow with chip-based electrophoresis. In this system, the dialysis probe is integrated with a PDMS chip that merges dialysate with fluorogenic reagent and segments the flow into 8–10 nL plugs at 0.3–0.5 Hz separated by perfluorodecalin. The plugs flow to a glass chip where they are extracted to an aqueous stream and analyzed by electrophoresis with fluorescence detection. The novel extraction system connects the segmented flow to an electrophoresis sampling channel by a shallow and hydrophilic extraction bridge that removes the entire aqueous droplet from the oil stream. With this approach, temporal resolution was 35 s and independent of distance between sampling and analysis. Electrophoretic analysis produced separation with 223,000 ± 21,000 theoretical plates, 4.4% RSD in peak height, and detection limits of 90–180 nM for six amino acids. This performance was made possible by three key elements: 1) reliable transfer of plug flow to a glass chip; 2) efficient extraction of aqueous plugs from segmented flow; and 3) electrophoretic injection suitable for high efficiency separation with minimal dilution of sample. The system was used to detect rapid concentration changes evoked by infusing glutamate uptake inhibitor into the striatum of anesthetized rats. These results demonstrate the potential of incorporating segmented flow into separations-based sensing schemes for studying chemical dynamics in vivo with improved temporal resolution.
PMCID: PMC2784254  PMID: 19803495
Microdialysis; segmented flow; temporal resolution; electrophoresis; amino acids
23.  Review of recent advances in analytical techniques for the determination of neurotransmitters 
Analytica chimica acta  2009;653(1):1-22.
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.
PMCID: PMC2759352  PMID: 19800472
neurotransmitter; biosensor; microdialysis
24.  Context-Independent, Temperature-Dependent Helical Propensities for Amino Acid Residues 
Journal of the American Chemical Society  2009;131(36):13107-13116.
Assigned from data sets measured in water at 2, 25, and 60 °C containing 13C=O NMR chemical shifts and [θ]222 ellipticities, helical propensities are reported for the twenty genetically coded amino acids, as well as for norvaline and norleucine. These have been introduced by chemical synthesis at central sites within length-optimized, spaced, solubilized Ala19 hosts. The resulting polyalanine-derived, quantitative propensity sets express for each residue its temperature-dependent but context-independent tendency to forgo a coil state and join a preexisting helical conformation. At 2 °C their rank ordering is: P ⪡ G < H < C, T, N < S < Y, F, W < V, D < K < Q < I < R, M < L < E < A; at 60 °C the rank becomes: H, P < G < C < R, K < T, Y, F < N, V < S < Q < W, D < I, M < E < A < L. The ΔΔ G values, kcal/mol, relative to alanine, for the cluster T, N, S, Y, F, W, V, D, Q, imply that at 2 °C all are strong breakers: ΔΔ Gmean = +0.63 ± 0.11, but at 60 °C their breaking tendencies are dramatically attenuated and converge toward the mean: ΔΔ Gmean = +0.25 ± 0.07. Accurate modeling of helix-rich proteins found in thermophiles, mesophiles, and organisms that flourish near 0 °C thus requires appropriately matched propensity sets. Comparisons are offered between the temperature-dependent propensity assignments of this study and those previously assigned by the Scheraga group; the special problems that attend propensity assignments for charged residues are illustrated by lysine guest data; and comparisons of errors in helicity assignments from shifts and ellipticity data show that the former provide superior precision and accuracy.
PMCID: PMC2770013  PMID: 19702302
25.  Continuous Operation of Microfabricated Electrophoresis Devices for 24 Hours and Application to Chemical Monitoring of Living Cells 
Analytical chemistry  2009;81(16):6837-6842.
Microchip electrophoresis is an emerging analytical technology with several useful attributes including rapid separation time, small sample requirements, and automation. In numerous potential applications, such as chemical monitoring or high-throughput screening, it may be desirable to use a system for many analyses without operator intervention; however, long term operation of microchip electrophoresis systems has received little attention. We have developed a microchip electrophoresis system that can automatically inject samples at 6 s intervals for 24 h resulting in collection of 14,400 assays in one session. Continuous operation time of a prototype of the device was limited to 2 h due to degradation of reagents and electrophoresis buffers on the chip; however, modification so that all reagents were continuously perfused into reservoirs on the device ensured fresh reagents were always used for analysis and enabled extended operating sessions. The electrophoresis chip incorporated a cell perfusion chamber and reagent addition channels to allow chemical monitoring of fluid around cells cultured on the chip by serial electrophoretic immunoassays. The immunoassay had detection limits of 0.4 nM for insulin and generated ∼4% relative standard deviation over an entire 24 h period with no evidence of signal drift. The combined system was used to monitor insulin secretion from single islets of Langerhans for 6 to 39 h. The monitoring experiments revealed that islets have secretion dynamics that include spontaneous oscillations after extended non-oscillating periods and possible ultradian rhythms.
PMCID: PMC2846223  PMID: 19621896

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