Search PubMed⌕ Search

Biomedical subjects

C E Lunte

Publications and source records attributed to C E Lunte.

At least 37 records · Page 2Linked to original sources
PubMed1998

Monitoring in situ liver metabolism in rats using microdialysis. Comparison of microdialysis mass-transport model predictions to experimental metabolite generation data.

The generation of metabolites from two model compounds, phenacetin and acetaminophen, included in the perfusion fluid of a microdialysis probe implanted into rat liver was studied. When 60 microM phenacetin was included in the perfusion fluid using a flow rate of 1.0 microL/min, acetaminophen and acetaminophen sulfate were recovered at concentrations that ranged between 0.4 and 1.6 microM. Acetaminophen sulfate ([AS]gain) diffused back into the microdialysis probe on a micromolar percentage basis of 8.9+/-2.4% (n = 3) when acetaminophen was passed through the probe at a concentration between 11 and 12 microM. When 220-240 microM acetaminophen was passed through the probe, the percentage of acetaminophen sulfate recovered was 4.8+/-1.4% (n = 3) (P < 0.1 compared to the 11 microM group). No acetaminophen glucuronide was detected in the dialysate samples. A mathematical model that describes mass transport in microdialysis sampling was used to predict the concentration of metabolite that could be recovered into the dialysate after the loss of a substrate compound that undergoes metabolism. The model predicts a metabolite recovery of 23.6% using estimates for phenacetin metabolism and 21.5% using estimates for acetaminophen metabolism. The results presented here indicate that microdialysis has potential to be used to study local in situ metabolism and with further refinements of the microdialysis mass-transport model may be used to estimate in vivo metabolic formation rates.

Acetaminophen↗
PubMed1997

Voltammetric detection for capillary electrophoresis.

Several approaches to implementing amperometric detection for capillary electrophoresis have been reported. This report describes the development of a voltammetric detector for CE. The detector is designed to minimize distortion of the voltammetry due to ohmic potential drop. This was accomplished by using a cast Nafion detection cell at the end of the separation capillary. The cast Nafion detection cell provided a low-dead-volume, low-resistance cell that minimized ohmic potential drop and peak band broadening. The ability to detect the current due to oxidation of analytes superimposed on a large background current was also improved. A dynamic background subtraction scheme was used in which a second working electrode, positioned in the electrochemical cell but outside of the detection cell, was used to compensate for the background current in real time. The output of the compensating working electrode was subtracted from the output of the detecting working electrode prior to analog-to-digital conversion. Postexperimental digital background subtraction was also implemented. This approach provided optimal elimination of the background current with maximal detection of the analytical signal. The voltammetric detector developed produced high-quality voltammetric response of analytes with injected concentrations as low as 0.20 microM. The system was evaluated by obtaining CE voltammograms of a mixture of eight test phenolic acids.

Electrodes↗
PubMed1997

Determination of caffeine and its metabolites by micellar electrokinetic capillary electrophoresis.

The determination of caffeine and its analogues is important for a wide variety of analyses and is performed in an assortment of matrices ranging from food to clinical samples. While reversed-phase HPLC has become the standard analysis protocol in most laboratories, capillary electrophoresis has the advantages of higher separation efficiency and shorter separation time. The micellar capillary electrophoresis (MECC) separation of caffeine and its metabolites, theobromine, paraxanthine, theophylline and 1,3,7-trimethyluric acid was investigated using sodium dodecyl sulphate (SDS) as the micellar phase. The effects of pH, micelle concentration, buffer concentration, ionic strength, buffer salts, applied voltage and injection time were studied to select the optimum conditions for the determination of caffeine and its four analogues in drugs, foods and body fluids. Caffeine and its three analogues were resolved within 120 s with detection limits less than 1 microgram/ml. Samples could be analyzed utilizing direct injection with satisfactory resolution and reproducibility.

Animals↗
PubMed1997

Simultaneous determination of the elimination profiles of the individual enantiomers of racemic isoproterenol using capillary electrophoresis and microdialysis sampling.

Microdialysis sampling and capillary electrophoresis with electrochemical detection (CE-EC) were used in combination to simultaneously define the elimination profile of each enantiomer of isoproterenol (ISP) administered as a racemic mixture to Sprague-Dawley rats. Resolution of the enantiomers of ISP was accomplished using a running buffer containing methyl-O-beta-cyclodextrin as a chiral recognition reagent. The CE-EC system provided a concentration limit of detection of 0.63 ng ml-1, allowing monitoring of the elimination of ISP for up to six half-lives. Microdialysis sampling was capable of continuously monitoring the concentration of ISP with 60 s resolution. The concentration versus time data for the elimination of (+) and (-) ISP were fit to a biphasic first order elimination model yielding average apparent distribution half-lives of 0.52 +/- 0.07 min and 0.55 +/- 0.08 min and average apparent elimination half-lives of 9.8 +/- 2.2 and 8.8 +/- 2.0 min for (-) and (+) ISP, respectively (n = 3 rats). No statistically significant difference in the average half-lives was found. However, because each enantiomer was simultaneously determined in each animal a paired two-sample t-Test could also be done. This statistical analysis demonstrated that a difference in the elimination half-lives of the enantiomers of ISP does exist.

Adrenergic beta-Agonists↗
PubMed1997

Factors that influence microdialysis recovery. Comparison of experimental and theoretical microdialysis recoveries in rat liver.

Inhibition of metabolic processes was used to assess the possible change in the recovery of material from a microdialysis probe implanted in vivo in rat liver. Phenacetin and antipyrine were perfused through a microdialysis probe implanted in the liver. Inhibition of phenacetin and antipyrine metabolism was achieved through an iv bolus dose of the cytochrome P450 suicide substrate 1-aminobenzotriazole (1-ABT). 1-ABT inhibited phenacetin clearance by 90%, thus also inhibiting metabolism by 90%. There was no statistical difference in the recovery of phenacetin and antipyrine across the microdialysis membrane in the liver between the control and metabolically inhibited animals. Partial differential equations were developed that describe the transport of analyte from the microdialysis probe and solved by an implicit finite-difference method to aid in the understanding of the above-mentioned microdialysis experiments. Predictions of microdialysis recovery obtained from the numerical model are compared with those found experimentally. The model could predict trends in the data, but not the actual experimental values. This suggests that predictions from this microdialysis model are essentially heuristic and as presently formulated can be used only to show mechanisms that affect recovery, but they cannot be used to accurately predict recovery. Prediction of actual recovery requires knowledge of the values of the parameters that describe chemical properties such as the in vivo diffusion coefficient, metabolism rate constant, and capillary exchange rate constant. For microdialysis experiments performed in the liver, capillary exchange and the rate of liver blood flow appear to be the dominant processes that facilitate net transport from a microdialysis probe rather than metabolic processes. These results indicate that microdialysis recoveries measured after inhibition of a concentration-dependent kinetic process via pharmacological challenge will change only when the kinetic process that is being challenged is large compared to the contribution of all concentration-dependent kinetic processes, including other metabolism routes, capillary exchange, or uptake that remove the analyte from the tissue space. It is concluded that the microdialysis recovery of a substance from the liver is not generally affected by liver metabolism.

Animals↗
PubMed1996

Optimization of the separation and detection of the enantiomers of isoproterenol in microdialysis samples by cyclodextrin-modified capillary electrophoresis using electrochemical detection.

Isoproterenol is a chiral catecholamine with a half-life of elimination of less than 10 min. In order to study the pharmacokinetics of this compound using microdialysis sampling, an analytical method was needed which could resolve the individual enantiomers of isoproterenol and required less than 1 microliter of sample. A capillary electrophoretic method using a run buffer containing methyl-O-beta-cyclodextrin as a chiral recognition agent was developed which could resolve the enantiomers of isoproterenol. The detection limits using UV absorbance detection were found to be too high to determine the concentration of isoproterenol in plasma for a sufficient time following administration to establish the pharmacokinetics. The detection limits were decreased three orders of magnitude to 3 ng/ml by using an amperometric detector. The detection limits were decreased to 0.6 ng/ml using an on-column concentration technique in which peak stacking was accomplished by following the sample injection with a plug of acid.

Animals↗
PubMed1996

Simultaneous microdialysis sampling from multiple sites in the liver for the study of phenol metabolism.

Microdialysis probes of the linear design were implanted in three regions of the liver of anesthetized rats. Probes planted 1 cm apart exhibited no cross communication as demonstrated by continuously perfusing one probe with phenol for over 2 hours while monitoring the other probes for phenol and its metabolites. Regional differences in metabolic profile were observed following intravenous administration of phenol. Significantly lower concentrations of the phase two metabolites, phenol-glucuronide and hydroquinone-glutathione conjugate, were found at the probe implanted at the anterior position relative to the probes at the median and posterior positions. The kinetics of elimination were not significantly different at any of these positions. Phenol was also directly delivered to the liver through the microdialysis probes. No differences in delivery of phenol or formation of metabolites were observed at the three regions.

Animals↗
PubMed1995

A perfluorosulfonated ionomer end-column electrical decoupler for capillary electrophoresis/electrochemical detection.

An end-column electrical decoupler contructed with perfluorosulfonated ionomer (Nafion) is described. This decoupler was fabricated at the cathodic end of the separation capillary by casting liquid Nafion ion exchange powder over a copper-plated tungsten wire. The internal diameter of the flow channel was controlled by adjusting the thickness of the copper plating. This design overcomes problems of conventional end-column detection such as low sensitivity due to low collection efficiency of analytes at the detection electrode, difficulty in precise placement of the detection electrode, and the need to use small-bore capillaries (<-25 micron). The loss of cationic analytes observed with long-cast Nafion on-column decouplers was significantly reduced. The high current shunting capability of the long Nafion decoupler was maintained in this configuration. Elimination of the detection capillary required for on-column electrical decouplers provided higher separation efficiency by maintaining plug-type flow throughout the system. Four model catecholamines were well separated within 5 min with separation efficiency of up to 230 000 plates and migration time reproducibilities of <0.6% RSD. With the optimized experimental conditions, detection limits of 3 nM for the catecholamines were achieved in a Ringer's solution matrix (to model a microdialysis sample).

Catecholamines↗
PubMed1995

Microdialysis sampling coupled on-line to fast microbore liquid chromatography.

A system which combines intravenous microdialysis sampling on-line with fast microbore liquid chromatographic analysis is described. Using a 14 mm x 1 mm I.D. ODS column, caffeine, theobromine and paraxanthine could be resolved in under 1 min and acetaminophen and its two main metabolites, the glucuronide and sulfate conjugates, could be resolved in 30 s. This fast separation allowed on-line microdialysis sampling to be conducted with a 1-min sampling interval. The system was evaluated by monitoring the pharmacokinetics following intravenous administration of acetaminophen and caffeine.

Acetaminophen↗
PubMed1995

Continuous in vivo monitoring of amino acid neurotransmitters by microdialysis sampling with on-line derivatization and capillary electrophoresis separation.

A separation-based biosensor has been developed that is capable of near-real-time analysis of aspartate and glutamate with a temporal resolution of less than 2 min in anesthetized or awake, freely moving animals. The instrument consists of a microdialysis sampling system, an on-line reactor, an injection interface, and a CE-LIF system. Primary amine analytes are derivatized with NDA/CN following microdialysis sampling using an on-line reactor to produce fluorescent CBI derivatives. The reaction takes approximately 1 min. The derivatized sample then travels to a microinjection valve which alternately sends CE running buffer and reacted microdialysis sample to the CE column via an injection interface. The interface allows a controllable volume of 10-20 nL to be injected onto the CE separation capillary. Separation of aspartate and glutamate from the other amino acids present in the microdialysis sample was achieved within 70 s. Detection limits for glutamate and aspartate using laser-induced fluorescence detection were 0.1 microM. The linear dynamic range was acceptable for the determination of aspartate and glutamate in dialysate samples where the levels are between 1 and 10 microM. Full automation of the system was achieved by computer control of the valve, the interface, and the data collection system. The performance of this system was demonstrated in an anesthetized rat by monitoring ECF levels of aspartate and glutamate released in brain after stimulation with high concentrations of K+.

Animals↗
PubMed1995

Microdialysis sampling coupled to on-line microbore liquid chromatography for pharmacokinetic studies.

Microdialysis sampling has been coupled on-line to microbore liquid chromatography for pharmacokinetic investigations in awake, freely-moving animals. Valve systems are described which provide continuous collection of the microdialysis sample for injection into the chromatographic system. One valve system consisted of two six-port valves used in tandem. The other valve system consisted of an eight-port valve with two sample loops. The eight-port valve system provided better precision than the dual six-port valve system yet worse than an autosampler. However, when used for dialysis samples, the precision of the various injection methods was equivalent. The on-line system was capable of rapidly following changes in the dialysate concentration and provided near real-time analysis of the plasma concentration of analytes. The system was evaluated by monitoring the pharmacokinetics of acetaminophen in awake, freely-moving rats.

Animals↗
PubMed1995

Dual-electrode amperometric detection for the determination of SR4233 and its metabolites with microbore liquid chromatography.

3-Amino-1,2,4-benzotriazine-2,4-di-N-oxide (SR4233) is a promising new antineoplastic agent based on reductive activation. SR4233 and its major metabolites (SR4317 and SR4330) are all easily reduced at a carbon electrode. Reductive amperometric detection can therefore provide high selectivity and low detection limits with chromatographic analysis and is an ideal approach to detection of SR4233 in microdialysis samples. However, in order to use amperometric detection in the reductive mode, sample deoxygenation is necessary. This is typically done by purging the sample with either argon or nitrogen prior to injection. This approach is not feasible for microdialysis samples because only 5-10 microliters is usually available. In this report, a microbore liquid chromatographic method with dual-electrode amperometric detection is described for the determination of SR4233 and its metabolites without predeoxygenation. A dual-electrode amperometric detector was used in the series configuration with an upstream potential of -450 mV to reduce SR4233 and its metabolites to a common product and a downstream potential of +400 mV to oxidize this product. Oxygen is only electroactive at the upstream electrode because of its irreversible behavior. This method is compatible with the small sample volumes provided by microdialysis sampling. Linear calibration graphs were obtained up to 55 microM for SR4233, and 140 microM for both SR4317 and SR4330. The detection limits were 70 nM for SR4233, and 50 nM for SR4317 and SR4330. The average intra-day variation over 5 days was 1.8% (SR4233), 1.4% (SR4330), and 1.8% (SR4317), whereas the inter-day variation over 5 days was 14.1% (SR4233), 8.6% (SR4317), and 2.6% (SR4330).

Animals↗
PubMed1995

Automated analytical systems for drug development studies. Part IV. A microdialysis system to study the partitioning of lomefloxacin across an erythrocyte membrane in vitro.

An automated system utilizing microdialysis sampling, intermittent dosing, and liquid chromatographic analysis was assembled in order to study the partitioning of lomefloxacin, a fluoroquinolone antimicrobial, into human erythrocytes in vitro. The apparent erythrocyte:buffer partition coefficient was found to be approximately 2.0 with this system and by a manual method. The value was concentration-dependent; lower partition coefficients were observed at lomefloxacin concentrations less than 1 microgram ml-1. At all concentrations, values obtained by microdialysis were statistically indistinguishable from those obtained by a conventional manual method. The results indicate that erythrocyte partition coefficients can be measured successfully with the microdialysis system. Furthermore, microdialysis sampling eliminates the tedious methodology associated with traditional erythrocyte partitioning studies, including sample clean-up. Due to automated intermittent dosing and on-line LC analysis, the system operates unattended.

Adult↗
PubMed1995

Microdialysis sampling for hepatic metabolism studies. Impact of microdialysis probe design and implantation technique on liver tissue.

Microdialysis sampling of liver tissue was performed using several probe geometries. The extent of tissue damage and response in vivo caused by implantation and indwelling of the probe was evaluated by histological examination of the tissue. A linear probe, implanted using fused silica tubing, was less damaging than other probe designs and implantation procedures tested. A series of time points up to 48 hr after implantation ere histologically examined. Infiltration of inflammatory cells, predominantly polymorphonuclear leukocytes (PMNs), was evident adjacent to the probe membrane after approximately 8 hr. Mixed inflammatory infiltration, mainly PMNs but including some macrophages, was observed in tissue slices 18 hr after implantation. At 48 hr, the mixed inflammatory infiltration was still present, with some degeneration of PMNs. In implantations of longer than 12 hr, some necrosis appeared at eh implantation site. The rate of delivery of phenol via the probe was stable for at least 30 hr, despite changes in the surrounding tissue.

Animals↗
PubMed1994

Liquid chromatographic determination of tacrine and its metabolites in rat bile microdialysates.

A microbore liquid chromatographic method using a phenyl stationary phase was developed for the determination of 9-amino-1,2,3,4-tetrahydroacridine (tacrine, THA) and its metabolites in microdialysis samples of bile. Analysis of microdialysis samples requires analytical methods with low detection limits and small sample volume requirements. The method uses a 1-mm I.D. phenyl column and fluorescence detection. A detection limit of 0.25 ng/ml in a 5-microliters sample was achieved for THA. This method was then used to determine THA and THA-1-ol in the bile dialysate of a rat. Because of the small sample volume requirements, a 10-min temporal resolution was achieved for the microdialysis experiment. The low detection limit allowed the THA concentration in the bile to be monitored for more than 4 h following a 1.0 mg/kg i.v. dose of THA.

Animals↗