To describe the objective sleep of patients receiving chemotherapy for multiple myeloma (MM) prior to stem cell transplantation.
A descriptive study with repeated measures.
An international referral center in an urban area of the southern United States.
A convenience sample of a subset of 12 patients with MM, recruited from a randomized, controlled trial.
Objective sleep was assessed using two nights of polysomnography, one obtained before and one after a second cycle of high-dose chemotherapy prior to stem cell transplantation. Demographic and clinical data were obtained through a retrospective chart review.
Main Research Variables
Objective sleep including sleep characteristics, sleep-related respiratory events, and periodic limb movements (PLMs) of sleep.
Sleep was characterized by a relatively short sleep time, excessive time spent awake after the onset of sleep, and poor sleep efficiency (objective sleep quality). Patients spent more than the expected percent of time in non–rapid eye movement sleep and less in rapid eye movement sleep. Arterial oxyhemoglobin saturation nadirs reflected episodes of low arterial oxygen saturation. PLMs during sleep were in the mildly elevated range.
Findings suggest that patients had poor sleep efficiency (objective sleep quality) and were slightly better sleepers after receiving a second cycle of high-dose chemotherapy. A number of patients also demonstrated obstructive sleep apnea and frequent PLMs.
Implications for Nursing
Findings support the need for additional investigation of sleep in patients with MM, particularly poor sleep efficiency and PLMs. Improving sleep may improve quality of life by decreasing associated symptoms such as pain, fatigue, and depression.
Oncology nurses should consider assessing patients with MM for insomnia symptoms, excessive daytime sleepiness, obstructive sleep apnea, and a history of jerking or kicking their legs when asleep. Those symptoms may suggest the need for additional investigation of a possible sleep disorder, which may negatively influence mood and function.
The ventrolateral thalamus (VL) is a primary relay point between the basal ganglia and the primary motor cortex (M1). Using dual probe microdialysis and locomotor behavior monitoring, we investigated the contribution of VL input into M1 during amphetamine (AMPH)-stimulated monoamine release and hyperlocomotion in rats. Tetrodotoxin (TTX) (10 uM) perfusion into the VL significantly lowered hyperactivity induced by AMPH (1 mg/kg i.p.). This behavioral response corresponded to reduced cortical glutamate and monoamine release. To determine which glutamate receptors the thalamocortical projections acted upon, we perfused either the AMPA/kainate receptor antagonist NBQX (10 μM) or the NMDA receptor antagonist (MK-801) intracortically followed by systemic AMPH. The results show that AMPA/kainate, and to a lesser extent NMDA receptors, mediated the observed effects. Since glutamate-monoamine interactions could possibly occur through local or circuit-based mechanisms, we isolated and perfused M1 tissue ex vivo to determine the extent of local glutamate-dopamine interactions. Taken together, these results demonstrate that AMPH generates hyperlocomotive states via thalamocortical signaling and that cortical AMPA receptors are an important mediator of these effects.
dopamine; microdialysis; glutamate; amphetamine; thalamus; motor cortex
Measurement of neuropeptides in the brain through in vivo microdialysis sampling provides direct correlation between neuropeptide concentration and brain function. Capillary liquid chromatography-multistage mass spectrometry (CLC-MSn) has proven effective at measuring endogenous neuropeptides in microdialysis samples. In the method, microliter samples are concentrated onto nanoliter volume packed beds before ionization and mass spectrometry analysis. The long times required for extensive preconcentration present a barrier to routine use because of the many samples that must be analyzed and instability of neuropeptides. In this study, we evaluated the capacity of 75 µm inner diameter (I.D.) capillary column packed with 10 µm reversed phase particles for increasing the throughput in CLC-MSn based neuropeptide measurement. Coupling a high injection flow rate for fast sample loading/desalting with a low elution flow rate to maintain detection sensitivity, this column was reduced analysis time from ~30 min to 3.8 min for 5 µl sample, with 3 pM limit of detection (LOD) for enkephalins and 10 pM LOD for dynorphin A1–8 in 5 µl sample. The use of isotopic labeled internal standard lowered peptide signal variation to less than 5%. This method was validated for in vivo detection of Leu and Met Enkephalin with microdialysate collected from rat globus pallidus (GP). The improvement in speed and stability makes capillary LC-MSn measurement of neuropeptides in vivo more practical.
Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound to free ratio. The method was used to screen a library of 3,443 compounds and results compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that reconfirmed in subsequent testing suggesting greater specificity. This finding was attributed to use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens but at the current stage of development it is attractive as a secondary screen to test hits found by higher throughput methods.
Mapping chemical dynamics in the brain of live subjects is a challenging but highly rewarding goal because it allows neurotransmitter fluctuations to be related to behavior, drug effects, and disease states. A popular method for such measurements is microdialysis sampling coupled to analytical measurements. This method has become well-established for monitoring low molecular weight neurotransmitters, metabolites, and drugs, especially in pharmacological and pharmacokinetic studies. Recent technological developments which improve the temporal and spatial resolution of the methods will enable it to be used for studying behavior and small brain nuclei. Better assays allow monitoring more neurotransmitters simultaneously. Extension to analysis of aggregating proteins like amyloid β are proving extremely useful for uncovering the roles of these molecules and how they contribute to neurodegenerative diseases.
2-bromohexadecanoic acid, or 2-bromopalmitate, was introduced nearly 50 years ago as a non-selective inhibitor of lipid metabolism. More recently, 2-bromopalmitate re-emerged as a general inhibitor of protein S-palmitoylation. Here, we investigate the cellular targets of 2-bromopalmitate through the synthesis and application of click-enabled analogues. In cells, 2-bromopalmitate is converted to 2-bromopalmitoyl-CoA, although less efficiently than free palmitate. Once conjugated to CoA, probe reactivity is dramatically enhanced. Importantly, both 2-bromopalmitate and 2-bromopalmitoyl-CoA label DHHC palmitoyl acyl transferases (PATs), the enzymes that catalyze protein S-palmitoylation. Mass spectrometry analysis of enriched 2-bromopalmitate targets identified PAT enzymes, transporters, and many palmitoylated proteins, with no observed preference for CoA-dependent enzymes. These data question whether 2-bromopalmitate (or 2-bromopalmitoyl-CoA) blocks S-palmitoylation by inhibiting protein acyl transferases, or by blocking palmitate incorporation by direct covalent competition. Overall, these findings highlight the promiscuous reactivity of 2BP, and validate clickable 2BP analogues as activity-based probes of diverse membrane associated enzymes.
Patients with type 2 diabetes (T2D) often exhibit hyperglucagonemia despite hyperglycemia, implicating defective α-cell function. Although endoplasmic reticulum (ER) stress has been suggested to underlie β-cell dysfunction in T2D, its role in α-cell biology remains unclear. X-box binding protein 1 (XBP1) is a transcription factor that plays a crucial role in the unfolded protein response (UPR), and its deficiency in β-cells has been reported to impair insulin secretion, leading to glucose intolerance. To evaluate the role of XBP1 in α-cells, we created complementary in vivo (α-cell–specific XBP1 knockout [αXBPKO] mice) and in vitro (stable XBP1 knockdown α-cell line [αXBPKD]) models. The αXBPKO mice exhibited glucose intolerance, mild insulin resistance, and an inability to suppress glucagon secretion after glucose stimulation. αXBPKD cells exhibited activation of inositol-requiring enzyme 1, an upstream activator of XBP1, leading to phosphorylation of Jun NH2-terminal kinase. Interestingly, insulin treatment of αXBPKD cells reduced tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) (pY896) and phosphorylation of Akt while enhancing serine phosphorylation (pS307) of IRS1. Consequently, the αXBPKD cells exhibited blunted suppression of glucagon secretion after insulin treatment in the presence of high glucose. Together, these data indicate that XBP1 deficiency in pancreatic α-cells induces altered insulin signaling and dysfunctional glucagon secretion.
Auditory cues can create the illusion of self-motion (vection) in the absence of visual or physical stimulation. The present study aimed to determine whether auditory cues alone can also elicit motion sickness and how auditory cues contribute to motion sickness when added to visual motion stimuli. Twenty participants were seated in front of a curved projection display and were exposed to a virtual scene that constantly rotated around the participant's vertical axis. The virtual scene contained either visual-only, auditory-only, or a combination of corresponding visual and auditory cues. All participants performed all three conditions in a counterbalanced order. Participants tilted their heads alternately towards the right or left shoulder in all conditions during stimulus exposure in order to create pseudo-Coriolis effects and to maximize the likelihood for motion sickness. Measurements of motion sickness (onset, severity), vection (latency, strength, duration), and postural steadiness (center of pressure) were recorded. Results showed that adding auditory cues to the visual stimuli did not, on average, affect motion sickness and postural steadiness, but it did reduce vection onset times and increased vection strength compared to pure visual or pure auditory stimulation. Eighteen of the 20 participants reported at least slight motion sickness in the two conditions including visual stimuli. More interestingly, six participants also reported slight motion sickness during pure auditory stimulation and two of the six participants stopped the pure auditory test session due to motion sickness. The present study is the first to demonstrate that motion sickness may be caused by pure auditory stimulation, which we refer to as “auditorily induced motion sickness”.
Western blotting is a commonly used assay for proteins. Despite the utility of the method, it is also characterized by long analysis times, manual operation, and lack of established miniaturized counterpart. We report a new way to Western blot which addresses these limitations. In the method, sodium dodecyl sulfate (SDS)-protein complexes are separated by sieving electrophoresis in a microfluidic device or chip. The chip is interfaced to a moving membrane so that proteins are captured in discrete zones as they migrate from the chip. Separations of SDS-protein complexes in the molecular weight range of 11 to 155 kDa were completed in 2 min with 4 × 104 theoretical plates at 460 V/cm. Migration time and peak area relative standard deviations were 3–6% and 0.2% respectively. Detection limit for actin was 0.7 nM. Assays for actin, AMP-kinase, carbonic anhydrase, and lysozyme are shown to demonstrate versatility of the method. Total analysis time including immunoassay was 22–32 min for a single sample. Because processing membrane for immunoassay is the slow step of the assay, sequential injections from different reservoirs on the chip and capture in different tracks on the same membrane allow increased throughput. As a demonstration, 9 injections were collected on one membrane and analyzed in 43 min (~5 min/sample). Further improvements in throughput are possible with more reservoirs or parallel channels.
In vivo calibration of microdialysis probes is required
for interpreting measured concentrations. The most popular method
of in vivo calibration is no-net-flux (NNF), which requires infusing
several concentrations of neurotransmitters to determine in vivo recoveries
(extraction fraction or Ed) and extracellular
concentrations. A new method for in vivo calibration of microdialysis
of neurotransmitters using glutamate (GLU) and dopamine (DA) as model
analytes is reported. 13C6-DA and 13C5-GLU were perfused through microdialysis probes as internal
calibrators. Using liquid chromatography with mass spectrometry, it
was possible to distinguish the 13C-forms from the endogenous
forms of each neurotransmitter. Ed was
directly calculated by measuring the loss of the 13C-forms
during infusion. The measured endogenous 12C forms of the
neurotransmitters could be corrected for Ed to give calibrated extracellular concentrations in vivo. Retrodialysis
of stable-isotope-labeled (SIL) neurotransmitters gave Ed and extracellular concentrations of 13C5-GLU and 13C6-DA that matched no-net-flux
measurements; however, the values were obtained in a fraction of time
because no added measurements were required to obtain the calibration. Ed was reduced during uptake inhibition for GLU
and DA when measured by SIL retrodialysis. Because Ed is directly measured at each microdialysis fraction,
it was possible to monitor changes in Ed under transient conditions created by systemic injection of uptake
inhibitors. The results show that DA and GLU concentrations are underestimated
by as much as 50% if not corrected for Ed during uptake inhibition. SIL retrodialysis provides equivalent
information to NNF at much reduced time and animal use.
No-net-flux; dopamine; glutamate; retrodialysis; cocaine; nucleus accumbens
Microfabricated fluidic systems have emerged as a powerful approach for chemical analysis. Relatively unexplored is the use of microfabrication to create sampling probes. We have developed a sampling probe microfabricated in Si by bulk micromachining and lithography. The probe is 70 μm wide by 85 μm thick by 11 mm long and incorporates two buried channels that are 20 μm diameter. The tip of the probe has two 20 μm holes where fluid is ejected or collected for sampling. Utility of the probe was demonstrated by sampling from the brain of live rats. For sampling, artificial cerebral spinal fluid was infused in through one channel at 50 nL/min while sample was withdrawn at the same flow rate from the other channel. Analysis of resulting fractions collected every 20 min from the striatum of rats by liquid chromatography with mass spectrometry demonstrated reliable detection of 17 neurotransmitters and metabolites. The small probe dimensions suggest it is less perturbing to tissue and can be used to sample smaller brain nuclei than larger sampling devices, such as microdialysis probes. This sampling probe may have other applications such as sampling from cells in culture. The use of microfabrication may also enable incorporation of electrodes for electrochemical or electrophysiological recording and other channels that enable more complex sample preparation on the device.
Although populations of neurons are known to vary on
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.
Dopamine; glutamate; push−pull perfusion; microdialysis; spatial resolution; in vivo
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.
striatum; leu-enkephalin; glycopeptide; blood-brain barrier penetration; mass spectrometry
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.
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.
oxytocin; vasopressin; microdialysis; mass spectrometry; paraventricular nucleus; hypothalamus
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.
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.
lipids; GCxGC; insulin
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.
electrophoresis; immunoassay; cells on chips; microfluidics; glucagon
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.
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.
Beta Cell; Insulin Secretion; Intermediary Metabolism; Metabolism; Metabolomics
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.
Neurotransmitter; Microdialysis; Benzoylation; Liquid Chromatography; Mass Spectrometry
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.
Globus pallidus; dopamine; enkephalins; mass spectrometry; microdialysis; amphetamine
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.
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.
Behaving; Electrophoresis; Microdialysis; Offline analysis; Plug storage; Segmented flow; Spatial resolution; Temporal resolution
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.