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Julie A Stenken

Publications and source records attributed to Julie A Stenken.

16 recordsLinked to original sources

Microdialysis sampling extraction efficiency of 2-deoxyglucose: role of macrophages in vitro and in vivo.

Macrophages are a class of inflammatory cells believed to direct the outcome of device biocompatibility. Despite their relevance to implanted in vivo devices, particularly implanted glucose sensors, few studies have attempted to elucidate how these cells affect device performance. Microdialysis sampling probes were used to determine glucose uptake alterations in the presence of resting and activated macrophages in vitro. Significant differences for 2-deoxyglucose (2-DG) relative recovery at 1.0 microL/min were observed between resting (74 +/- 7%, n = 18) and lipopolysaccharide (LPS) (1 microg/mL)-activated (56 +/- 6%, n = 18) macrophages in culture that had 2-DG spiked into the media (p < 0.005). To establish if in vitro characterization could be correlated to in vivo studies, microdialysis probes were implanted into the dorsal subcutis of male Sprague-Dawley rats for 0, 3, 5, and 7 days. An internal standard, 2-DG, was passed through the microdialysis probe during in vivo studies. No significant differences in 2-DG extraction efficiency from the probe into the tissue site were observed in vivo among microdialysis probes implanted into the subcutaneous space of Sprague-Dawley rats for either 3, 5, or 7 days vs probes implanted the day of sample collection. These results suggest that macrophage activation in vivo at implant sites is much lower than highly activated macrophages in vitro. It is important to note that these results do not rule out the potential for increased glucose metabolism at sensor implant sites.

Animals↗

Microdialysis sampling membrane performance during in vitro macromolecule collection.

Microdialysis sampling is well-established for sampling small molecules. Recently, there has been an increased interest toward collecting macromolecules using microdialysis sampling. In this work, fluorescein isothiocyanate-labeled dextrans (FITC-dextrans) with molecular weight between 10 and 70 kDa were chosen as representative molecules to study analyte mass transport properties during microdialysis sampling using different lengths (2 and 10 mm) of 100-kDa MWCO polyethersulfone membranes. Experiments were performed in both well-stirred and quiescent phosphate-buffered saline solutions as well as in a 0.3% agar solution. Different fundamental parameters affecting microdialysis sampling of macromolecules, including effective membrane diffusion coefficients, were evaluated. The applicability of the most-often-cited Bungay et al. mass transfer model was compared to experimental data for the FITC-dextrans. For the larger macromolecules, the membrane provides a significant mass transport resistance most likely caused by hindered diffusion. These experimental aspects that are critical to microdialysis sampling of macromolecules are presented.

Dextrans↗

Natural and synthetic affinity agents as microdialysis sampling mass transport enhancers: current progress and future perspectives.

Microdialysis sampling is a diffusion-based separation method that allows analytes to freely diffuse across a hollow fiber semi-permeable dialysis membrane. This sampling technique has been widely used for in vivo chemical collection. The inclusion of affinity-based trapping agents into the microdialysis perfusion fluid serves to improve the relative recovery via the binding reaction of low molecular weight hydrophobic analytes and larger analytes such as peptides and proteins. Here, we briefly review our past studies using different compounds (native cyclodextrins and antibodies) to improve microdialysis sampling recovery. A brief compilation of our studies using antibody-immobilized beads as a means to improve cytokine collection during microdialysis sampling is also described. We present new work focused on the use of antibody-immobilized bead microdialysis sampling enhancement for various endocrine hormones (amylin, GLP-1, glucagon, insulin, and leptin). The antibody-bead enhancement approach allowed for recovery enhancements that ranged between 3 and 20-fold for these peptides. Using the enhanced recovery approach, endocrine peptides at pM concentrations can be quantified. Finally, our initial work focused on developing non-antibody based enhancement agents using bovine serum albumin-heparin conjugates covalently bound to polystyrene microspheres is presented for the cytokine, tumor necrosis factor-alpha (TNF-alpha). Unlike antibodies, heparin provides the advantage of being reusable as an enhancement agent and served to improve the relative recovery of TNF-alpha by three-fold.

Biosensing Techniques↗

Microdialysis sampling of cytokines.

Microdialysis sampling is a well-known method for collection of low molecular weight hydrophilic analytes. Due to the success of this sampling technique for these analytes, many researchers have wanted to extend the use of this method to a wider range of analytes-particularly proteins and peptides. These analytes pose unique challenges during microdialysis sampling. The primary challenges are the reduced recovery across the dialysis semi-permeable membrane and the volume limitations/requirements for the typical immunoassay methods used for detection of proteins. This review covers the practical and theoretical aspects needed for in vivo microdialysis sampling of cytokines, which are a vitally important class of signaling proteins. In addition to the basics of the microdialysis method for sampling cytokines, the use of the microdialysis device as a localized cytokine delivery method is also described. Since relative recovery of cytokines is often low during microdialysis sampling, methods to improve the membrane recovery are discussed for in vitro and in vivo applications.

Animals↗

No delayed temporal response to sample concentration changes during enhanced microdialysis sampling using cyclodextrins and antibody-immobilized microspheres.

The temporal response to concentration changes external to a microdialysis probe containing trapping agents in the perfusion fluid was studied. Native beta-cyclodextrin and a water-soluble beta-cyclodextrin polymer were used as trapping agents in the microdialysis perfusion fluid to study the temporal concentration response to carbamazepine, a hydrophobic analyte. The temporal response of microdialysis probes containing antibody-immobilized microspheres against five different cytokines (tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5) to concentration changes outside of the probe was also determined. In both cases, no delayed temporal response of enhanced microdialysis was observed for either carbamazepine or the cytokines as compared to standard microdialysis sampling procedures.

Animals↗

In vitro characterization of an in situ microdialysis sampling assay for elastase activity detection.

A microdialysis sampling method has been developed to detect the in vitro presence of a proteolytic enzyme, porcine elastase, external to a microdialysis probe. Elastase converts the substrate, succinyl(Ala)(3)-p-nitroanilide (suc(Ala)(3)-p-NA), to p-nitroaniline (p-NA). The substrate, suc(Ala)(3)-p-NA, was locally delivered through the microdialysis probe to external solutions containing different elastase activities (0.025-0.5 units/mL). The product, p-NA, was recovered back into the probe. Dialysates containing both suc(Ala)(3)-p-NA and p-NA were quantified using HPLC-UV. Different microdialysis suc(Ala)(3)-p-NA extraction efficiencies (EE) were observed among different elastase-containing solutions (buffer and 0.3% agar solutions). The p-NA concentrations recovered back into the microdialysis probe correlated with the elastase activity external to the microdialysis probe. The greatest fraction of p-NA recovered as compared to substrate lost occurred with the highest flow rate used (5.0 microL/min). However, the highest concentrations of p-NA recovered occurred at the lowest flow rates. This method may allow for microdialysis sampling to be used as a means to study localized enzyme activity.

Agar↗

Multiplexed cytokine detection in microliter microdialysis samples obtained from activated cultured macrophages.

Microdialysis sampling probes were used to collect cytokine samples from lipopolysaccharide (LPS)-stimulated macrophages. The probes were immersed into cell culture wells containing either RAW 264.7 or isolated peritoneal macrophages. Dialysates (15 microL) from these wells were subjected to a multiplexed cytokine sandwich immunoassay platform analyzed by flow cytometry that measures up to six separate cytokines, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interferon-gamma (IFN-gamma), macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha) in a single 15-muL sample. In vitro microdialysis sampling relative recovery experiments showed that only IFN-gamma, IL-6, MCP-1, and TNF-alpha could be recovered across a commercially-available 100-kDa MWCO microdialysis membrane. Eleven hours after LPS addition (1 microg/mL), RAW 264.7 macrophages secreted greater than 8000 pg/mL of TNF-alpha and greater than 1000 pg/mL MCP-1. With the peritoneal macrophages, greater than 6000 pg/mL of IL-6, MCP-1, and TNF-alpha were obtained. The maximum dialysate concentrations obtained from the RAW macrophages were 1300 pg/mL for TNF-alpha and 55 pg/mL for MCP-1. Maximum cytokine concentrations from peritoneal macrophage dialysates reached approximately 2000 pg/mL, 1100 pg/mL and 500 pg/mL for TNF-alpha, MCP-1 and IL-6, respectively. Microdialysis sampling allowed for 20-min samples to be collected during the cytokine release from the activated macrophages. These results demonstrate that microdialysis sampling can be used for collection of selected cytokines with improved temporal resolution.

Animals↗

In vivo microdialysis sampling of cytokines produced in mice given bacterial lipopolysaccharide.

Cytokines are proteins that mediate communication between cells of the immune system as well as certain other non-immune host cells. These proteins are produced by many cell types and they mediate immune and inflammatory responses. However, the direct site analysis of these critical proteins is hampered by the lack of site-specific tools available for such direct measurements. In this study, both in vitro and in vivo microdialysis sampling of different cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], interleukin-6 [IL-6], IL-12p70, and macrophage chemoattractant protein-1 [MCP-1]) was performed. A mouse model of bacterial lipopolysaccharide (LPS) administration and response pattern was used for in vivo studies. Three cytokines, TNF-alpha, IL-6, and MCP-1 were quantified in the serum from mice given LPS. In vivo studies demonstrated the ability to monitor increasing levels of these cytokines (TNF-alpha, IL-6, and MCP-1) via microdialysis probes placed in the peritoneal cavity of mice given LPS. All three cytokines were quantified simultaneously in 15 muL of dialysate using a multiplexed bead-based immunoassay for flow cytometry. The detected dialysate cytokine concentrations varied between 200 pg/mL and 1500 pg/mL for TNF-alpha, between 600 pg/mL and 3000 pg/mL for MCP-1, and between 2700 pg/mL and more than 5000 pg/mL for IL-6. The detected serum cytokine concentrations ranged from 5700 pg/mL to 35,000 pg/mL for TNF-alpha, from 40,000 pg/mL to 65,000 pg/mL for MCP-1, and greater than than 100,000 pg/mL for IL-6. This work demonstrates that microdialysis sampling can be used in vivo to collect temporal profiles of cytokine production.

Animals↗

Microdialysis sampling combined with electron spin resonance for superoxide radical detection in microliter samples.

Quantitation of superoxide radical (O2.-) production at the site of radical generation remains challenging. Microdialysis sampling is an advantageous tool for sampling from localized environments. It is difficult to combine electron spin resonance (ESR) spin traps with microdialysis because O2.- adducts with common nitrone spin traps have shorter half-lives than typical microdialysis collection times. Furthermore, typical dialysate samples (5-15 microL) suffer significant sensitivity loss when diluted for detection in a conventional ESR flat cell (200 microL). To overcome these difficulties, a cyclic hydroxylamine, 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H), which produces a stable nitroxide radical (PP.) product upon reaction with O2.- was employed. Capillary cells (1.4 microL effective volume) coupled with a loop-gap resonator were utilized to measure PP. in microliter microdialysis samples (LOD 0.36 pmol). A xanthine/xanthine oxidase (X/XO) model system provided sustained O2.- production. When PP-H was included in the X/XO medium external to the microdialysis probe, a relative recovery of 22.1 +/- 1.1 and 57.2 +/- 5.7% for PP. was achieved at perfusion fluid flow rates of 0.5 and 1.0 microL/min, respectively. The respiratory burst in interferon-gamma and zymosan-stimulated RAW 264.7 macrophages was also investigated.

Electron Spin Resonance Spectroscopy↗

Enhanced microdialysis relative recovery of inflammatory cytokines using antibody-coated microspheres analyzed by flow cytometry.

Achieving high relative recovery (RR) of proteins during microdialysis sampling is difficult due to the diffusion limitations inherent to this sampling process. This often causes low microdialysis RR for proteins with molecular weight >10 kDa. A RR enhancement process for microdialysis sampling of proteins has been developed that can be readily used with flow cytometers. Multiplexed RR enhancement and detection of five different cytokines (TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-5) was achieved by including antibody-coated microspheres in the microdialysis perfusion fluid. Inclusion of these antibody-coated microspheres causes an increase in the analyte diffusive driving force across the dialysis membrane and a subsequent increase in the relative recovery. For the five cytokines, typical control and enhanced relative recoveries at a 1.0 microL/min flow rate were as follows (n = 3): TNF-alpha, 5.5 +/- 0.6% and 60.4 +/- 5.8%; IFN-gamma, 2.6 +/- 0.3% and 25.8 +/- 2.3%; IL-5, 1.4 +/- 0.2% and 4.9 +/- 0.1%; IL-4, 10.9 +/- 0.6% and 78.8 +/- 8.0%; and IL-2, 4.1 +/- 0.4% and 19.8 +/- 2.5%. Using this approach, a four- to 12-fold enhancement of microdialysis RR was achieved for the five cytokines from a quiescent solution. The enhancement varies among the five cytokines and may be due to different diffusive and antibody binding properties. TNF-alpha exhibited the highest RR enhancement, while IL-5 exhibited the lowest. Experimental parameters that affect the enhancement, such as flow rate, sample collection volume, and bead density, were studied.

Antibodies↗

Liquid chromatographic aqueous product characterization of high-energy electron beam irradiated 2-chlorobiphenyl solutions.

Polychlorinated biphenyls (PCBs) are of environmental concern due to their toxic nature. Ionizing radiation has been suggested as a means to remediate PCB-contaminated samples in complex matrices. A set of experiments was performed to qualitatively and quantitatively determine the aromatic degradation products of 2-monochlorobiphenyl (2-MCB) in an aqueous system exposed to ionizing radiation. The degradation of 2-MCB was observed in aqueous samples that were exposed to radiation from a linear accelerator electron beam source. Analytical measurements performed by liquid chromatography (LC) equipped with an ultraviolet (UV) detector revealed that biphenyl, o-hydroxybiphenyl, p-hydroxybiphenyl, phenol, chlorobenzene, and other unidentified products were created after 2-MCB irradiation. These results suggest that sensitive and selective analytical methods will be required to account for all degradation products during ionizing radiation of aqueous PCB-contaminated samples.

Biphenyl Compounds↗

Improving microdialysis extraction efficiency of lipophilic eicosanoids.

Microdialysis recovery of the lipophilic analytes prostaglandin B2, leukotriene B4 and C4 was studied in vitro. Relative recovery (RR) through different commercially-available microdialysis probes for prostaglandin B2 and leukotrienes was examined using different flow rates. The enhancing effect at different concentrations of binding agents such as alpha, beta, gamma-cyclodextrins (alpha, beta, gamma-CD) on the microdialysis RR for different eicosanoids was evaluated. Small organic molecules such as ethanol, propylene glycol and dimethyl sulfoxide (DMSO) were studied in terms of their effect on enhancing RR. Inclusion of arachidonic acid in either the perfusion fluid or the sample medium caused the microdialysis RR for these hydrophobic analytes to be increased.

Chromatography, High Pressure Liquid↗

Quantitation of nitric oxide-derived nitrite from activated macrophages using microdialysis sampling.

An HPLC method for detecting nitrite in microdialysis samples obtained from activated RAW 264.7 macrophages in cell culture has been developed. Nitrite was quantified using a pre-column derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by HPLC-UV analysis of the azide product. For dialysates, the detection limit of nitrite was 750 nM and the quantitation limit was 2.5 microM. The microdialysis relative recovery of nitrite in the macrophage cell culture medium was determined to be 86+/-2% (n=3) at a flow rate of 0.7 microl/min. Nitrite produced from activated macrophages was measured immediately after lipopolysaccharide (LPS) stimulation using microdialysis sampling.

Animals↗

Water-soluble cyclodextrin polymers for enhanced relative recovery of hydrophobic analytes during microdialysis sampling.

Microdialysis relative recovery (RR) enhancement using different water-soluble, epichlorohydrin-based cyclodextrin polymers (CD-EPS) was studied in vitro for different analytes, amitryptiline, carbamazepine, hydroquinone, ibuprofen, and 4-nitrophenol. When compared to the native CDs (alpha, beta, and gamma) on a per mole basis, the CD-EPS enhanced microdialysis RR was either statistically greater or the same. beta-CD-EPS was more highly retained than native beta-CD by a 20 000 Da molecular weight cutoff (MWCO) polycarbonate membrane, but showed no statistical difference for loss across a 100 000 Da MWCO polyethersulfone membrane (PES). When the same weight percent of beta-CD or beta-CD-EPS was included in the microdialysis perfusion fluid, the beta-CD-EPS produced a higher microdialysis RR than native beta-CD for all analytes across the PES membrane. However, enhancements for the PC membrane were statistically insignificant when beta-CD and beta-CD-EPS were compared on a per mole basis. These results suggest that CD-EPS may be used as effective enhancement agents during microdialysis sampling and for some membranes provide the additional advantage of being retained more than native CDs.

Cellulose↗

Magnetic resonance imaging of a tissue/implanted device biointerface using in vivo microdialysis sampling.

Real-time in vivo images of magnetic resonance contrast agent diffusion from implanted microdialysis probes were obtained by magnetic resonance (MR) microscopy. A gadolinium-containing contrast agent (Gd-DTPA) was infused through microdialysis probes implanted into the subcutaneous space of male Sprague-Dawley rats. The infusion of Gd-DTPA alters the T1 relaxation time for water protons near the microdialysis probe, thus causing an increase in brightness around the probe. Steady state concentration profiles of Gd-DTPA around the microdialysis probe were attained within 10 min. The distance for the diffusion of Gd-DTPA away from the probe was calculated to be approximately 1400 microm on the basis of an image intensity analysis. A 5-cm field of view was used with a 256 x 256 matrix, giving a voxel volume of 0.190 mm3 (195 microm x 195 microm x 5,000 microm). These experiments demonstrate the ability of magnetic resonance microscopy to obtain real-time images of Gd-DTPA diffusion around implanted microdialysis probes. This noninvasive technique may be useful for determining how fibrous encapsulation during long-term implantation may affect localized mass transport at a biointerface.

Animals↗

An in vitro hydroxyl radical generation assay for microdialysis sampling calibration.

A xanthine oxidase hydroxyl radical (.OH)-generating system was created for sustained in vitro production of *OH. This assay was coupled with microdialysis sampling to elucidate the factors that influence microdialysis calibration during radical trapping. A *OH trapping agent, 4-hydroxybenzoic acid, was included either in the microdialysis perfusion fluid or in the medium external to the microdialysis probe. Xanthine oxidase enzymatic activity was reproducible and had an average activity measured by UV absorbance of produced uric acid of 0.037 +/- 0.005 deltaAU/min (n = 5). A considerable amount of variance in the rate and amount of the product, 3,4-dihydroxybenzoic acid (3,4-DHBA), was observed when one microdialysis probe was placed in the reaction mixture. When two microdialysis probes were placed in the reaction mixture, a greater rate and amount of 3,4-DHBA was observed. Different concentrations of 3,4-DHBA were obtained between quiescent and stirred systems.

Ascorbic Acid↗