A Review of Microdialysis Sampling Systems

The most important aspects of microdialysis are a theoretical understanding of the process, the microdialysis membrane and the design of the microdialysis probe including the inner cannula dimensions. Several efforts have been made to theoretically account for the processes that take place during microdialysis. These have been employed to develop optimal sampling conditions so as to increase the applicability of the technique for in situ sampling and as a sample clean-up technique prior to chromatography. On the occasion of Prof. Lo Gorton’s 60th birthday, this review highlights the challenge presented by low analyte recoveries that is the major bottleneck in the wider use of microdialysis. The discussion concludes by considering how to increase analyte recovery through a multiple probe approach or by an increase in recovery in the light of the advantages of nanotechnology. Both approaches could impact on the use of microdialysis as a sampling and sample clean-up technique for liquid chromatography.     

Diffusion and calibration properties of microdialysis sampling membranes in biological media

The diffusion and calibration properties for three commercially available microdialysis membranes (polycarbonate-polyether (PC), polyacrylonitrile (PAN), and cuprophan (CUP)) were evaluated. The analytes studied had molecular weights between 94 (phenol) and 1355 (vitamin B12). For each analyte-membrane pair, an effective membrane diffusion coefficient was calculated. Effective membrane diffusion coefficients varied considerably between the microdialysis membranes. For Vitamin B12, CUP and PAN membranes gave relative recovery values of greater than 20% at 0.5 microl min(-1), while the PC membrane had a 1% recovery. When backpressure was applied. PC and PAN membranes exhibited more ultrafiltration than CUP membranes. Ultrafiltration did not affect analyte relative recovery through either PC or PAN membranes. Effective membrane diffusion coefficients were not significantly altered for some membrane-analyte combinations when exposed to 4% bovine serum albumin or 0.3% fibrinogen. These data suggest that reductions in relative recovery during long-term microdialysis sampling experiments may be due to other physiologically relevant proteins or to tissue reactions near the dialysis membrane.     

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.     

Enhancing the microdialysis recovery for sampling of Cu and Ni by incorporating humic acid in the perfusion liquid

A strategy to enhance the microdialysis relative recovery for sampling of Cu and Ni ions is presented. Enhanced recovery was achieved by incorporating humic acid, a binding agent, in the microdialysis perfusion liquid during sampling from a Cu and Ni standard solution mixture. All microdialysis sampling experiments were carried out at room temperature under quiescent conditions using a concentric type of microdialysis probe with an adjustable effective dialysis length. For all metal determinations electrothermal atomic absorption spectrometry was employed. Metal recoveries were shown to be dependent on the membrane molecular weight cut-off, perfusion rate, sample solution pH, perfusion liquid composition as well as perfusion liquid pH. Complete recoveries (100%) of Cu and Ni were obtained by microdialysis sampling using a 10 kDa molecular weight cut-off polysulfone membrane at a flow-rate of 2 μl/min employing a 0.05% (w/v) optimal composition of humic acid incorporated in the perfusion liquid. The optimal sampling pH of humic acid was determined to be 6 where most oxygen containing functional groups are dissociated and available for metal binding. These data have important ramifications for sampling and determination of metal ions in small sample solutions (∼10 ml) at very low concentrations in the ppb range.     

Enhanced microdialysis recovery of some tricyclic antidepressants and structurally related drugs by cyclodextrin-mediated transport

The enhanced microdialysis relative recovery (RR) of some hydrophobic tricyclic drugs (imipramine, desipramine, amitriptyline, carbamazepine and promethazine) is discussed. Enhanced RR was achieved by including a binding agent [beta-cyclodextrin (beta-CD) or 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD)] in the microdialysis perfusion fluid to form inclusion complexes with the drugs, which increases the analyte flux through the membrane material. The maximum effect of the RR increase for all the drugs studied was observed using a commercially available polycarbonate-polyether (PC) membrane. With a 4 mm PC membrane and 4.41 mmol l-1 (0.5% w/v) beta-CD included in the microdialysis perfusion fluid (0.9% saline, pH 7.4) at a flow rate of 0.5 microliter min-1, RR enhancements over controls were as follows: carbamazepine 136, imipramine 268, desipramine 298, amitriptyline 634, and promethazine 987%. Increasing beta-CD [up to 17.63 mmol l-1 (2% w/v)] or HP-beta-CD [up to 32.5 mmol l-1 (5% w/v)] concentration in the microdialysis perfusion fluid enhanced carbamazepine RR three (beta-CD) to four (HP-beta-CD) times compared to controls through PC microdialysis membranes. The PC membrane gave enhanced RR values that were twice those for cuprophan or AN-69 membranes. Enhanced RR with cyclodextrins was successfully applied to sampling from a protein solution containing desipramine in a 4% w/v bovine serum albumin solution. These results suggest that addition of cyclodextrins to microdialysis perfusion fluids may be used to increase microdialysis RR during blood sampling.     

Enhancing microdialysis recovery of metal ions by incorporating poly-l-aspartic acid and poly-l-histidine in the perfusion liquid

A study of the evaluation of poly-l-aspartic acid and poly-l-histidine as binding agents to enhance microdialysis recovery of metal ions is presented. Investigations were carried out to compare microdialysis recovery for Cr, Cu, Ni, and Pb when using water as the perfusion liquid as well as when using various concentrations of poly-l-aspartic acid and poly-l-histidine in the perfusion liquid. All experiments were carried out under quiescent conditions using a concentric type of microdialysis probe fitted with a polysulfone membrane having a 30 kDa molecular weight cut-off and a 10 mm effective dialysis length. The metal ions were determined using an electrothermal atomic absorption spectrometer equipped with a Zeemann background corrector. Incorporation of 0.032% (w/v) of poly-l-aspartic acid enhanced the recovery of Cu and Pb by factors of 90 and 64%, respectively (%RSD<3). The recovery of Cr was enhanced by 5%, but that of Ni never exceeded values achieved using ultra pure water. The use of 20% (w/v) of poly-l-histidine resulted in enhancement factors of 66 and 4% for Cu and Pb, respectively (%RSD<2). For both Cr and Ni, the recovery never exceeded that achieved with water. The data from these studies demonstrate the suitability of poly-l-aspartic and poly-l-histidine as selective and effective binding agents that enhance the microdialysis recovery of metal ions. Application of the optimised conditions to the determination of Pb and Cu in a wastewater sample confirmed the versatility of microdialysis, as higher recoveries of Cu were obtained with poly-l-aspartic acid compared to direct determination.     

In vivo sampling using loop microdialysis probes coupled to a liquid chromatograph.

Microdialysis probes with longer membranes (20-100 mm) provide increased relative recovery over traditional shorter probes (1-4 mm) developed for neuroscience applications. The characterization and optimization of "straight through" or "loop type" probes for use in subcutaneous tissue are considered. Membrane area, probe size, inlet and outlet tubing dimensions, and flow-rate are examined for their effects on relative recovery, the total collection rate, and bulk flow through the membrane wall. Polyacrylonitrile and regenerated cellulose membrane fibers with different geometries were compared. Sampling probes used fibers 3-10 cm long. Inlet and outlet tubing was varied from 25 to 110 microns I.D. with lengths of 10 to 50 cm. Probe configurations optimized for relative recovery, flow-rate, and utility for in vivo use are presented. Utilizing microdialysis probes with large membrane surface areas results in relative concentration recovery of greater than 50% at flow-rates of greater than 5 microliters/min. Therapeutic drug monitoring in subcutaneous tissue of awake animals is explored.     

Application of residue distribution along the sequence for discriminating outer membrane proteins

Discriminating outer membrane proteins from other folding types of globular and membrane proteins is an important problem both for detecting outer membrane proteins from genomic sequences and for the successful prediction of their secondary and tertiary structures. In this work, we have systematically analyzed the distribution of amino acid residues in the sequences of globular and outer membrane proteins. We observed that the occurrence of two neighboring aliphatic and polar residues is significantly higher in outer membrane proteins than in globular proteins. From the information about the dipeptide composition we have devised a statistical method for discriminating outer membrane proteins from other globular and membrane proteins. Our approach correctly picked up the outer membrane proteins with an accuracy of 95% for the training set of 337 proteins. On the other hand, our method has correctly excluded the globular proteins at an accuracy of 79% in a non-redundant dataset of 674 proteins. Furthermore, the present method is able to correctly exclude alpha-helical membrane proteins up to an accuracy of 87%. These accuracy levels are comparable to other methods in the literature. The influence of protein size and structural class for discrimination is discussed.     

Contribution of cell-surface components to Cu2+ adsorption by Pseudomonas putida 5-x

The contribution of various cell-surface components to Cu²⁺ adsorption by a Gram-negative bacterium, Pseudomonas putida 5-x, that was isolated from local electroplating effluent with a high capability to accumulate heavy metal ions was studied. The cell superficial layer had a negative effect on Cu²⁺ adsorption of the bacterial cells. Cu²⁺ adsorption capacity of the separated cell envelopes was fivefold more than that of the intact cells, owing to the liberation of more and more binding sites during the separation process. Some main components in the cell envelope, such as the peptidoglycan (PEG) layer, outer membrane, and inner membrane, provide the capability for Cu²⁺ adsorption. The content of the components in the cell envelope is in the order inner membrane > outer membrane > PEG layer, and their Cu²⁺ adsorption capacity was in the order PEG layer > outer membrane > inner membrane. The total contribution of the separated PEG layer material to Cu²⁺ adsorption by the cell envelope was no more than 15%, and the outer membrane and inner membrane contributed about 30–35% and 25–30%, respectively. The relatively high phospholipid content in the outer membrane may be the major reason for the higher adsorption capacity of the outer membrane to Cu²⁺ and, hence, such a high Cu²⁺ adsorption capacity of P. putida 5-x cell envelope.     

Determination of fluoxetine and norfluoxetine in rat brain microdialysis samples following intraperitoneal fluoxetine administration

Fluoxetine (FLX) and the N-desmethyl metabolite, norfluoxetine (NFLX) in rat brain microdialysis samples were determined by high-performance liquid chromatography (HPLC) with fluorescence detection using pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). In vitro experiment showed that the relative recovery of FLX across microdialysis membrane was enhanced by adding β-cyclodextrin (β-CD) or β-CD polymer to microdialysis perfusion fluid. The perfusion fluid containing β-CD polymer, which has polymeric glyceryl linkers attached to the hydroxyl group, gave the better recovery with satisfactory precisions. Using 1% β-CD polymer in Ringer’s solution as the perfusate, in vivo rat brain microdialysis experiment on intraperitoneal administration of FLX (10 mg/kg) to rats was carried out. The fluorescence peaks of FLX and NFLX appeared later than 30 min after the administration of FLX, and then, gradually increased with time. Two hours later, FLX reached a plateau level, but NFLX slowly increased, and at 24-48 h, NFLX levels were higher than FLX levels. These data suggest that long distributions of FLX and the potent metabolite, NFLX, in brain contributed to the long-term drug actions in vivo.     

A London-type formula for the dispersion interactions of endohedral A@B systems

A London-type formula is derived for endohedral systems. It involves the static dipole polarisability, alpha(1)(A) of the inner system, A, and a new type of dipole polarisability, alpha(-2)(B) with an r(-2) radial operator, for the outer system, B. The new formula has no explicit dependence on the radius, R, of B. The predicted interaction energies are compared against MP2 supermolecular calculations for A@C(60), A = He-Xe, Zn, Cd, Hg, and CH(4).     

In vitro study of experimental factors affecting the microdialysis results

The aim of the present study was to determine the influence of a series of experimental conditions (probe, perfusate flow rate and the method used to ascertain recovery) as well as the pharmacokinetic variables (concentration and time) on the estimation of the recovery coefficient of microdialysis probes. Two in vitro pharmacokinetic assays were also carried out to compare the results provided by microdialysis and those obtained with traditional sampling. The probes used were made in our laboratory and ciprofloxacin was used as a model compound. The results revealed that in all cases recovery was dependent on the probe and independent of time for a 80 min sampling time period. The effects of concentration on recovery depend on the flow rate; this was not statistically significant for a flow rate of 2 μl/min but an increase in flow rate to 6 μl/min transformed this parameter into a concentration-dependent variable. A decrease in recovery parallel to the increase in flow rate was found, with an exponential relationship between the two variables. Statistically significant differences were also found between the recovery values obtained for direct dialysis (18.44±1.61) and retrodialysis by loss of the analyte of interest (16.79±3.42). The values of the protein binding of ciprofloxacin as calculated by equilibrium dialysis and by microdialysis were similar. Characterization of the in vitro kinetic profile revealed no statistically significant differences for coefficients and exponents obtained by traditional sampling and microdialysis, although the confidence intervals of the curves were wider for microdialysis.     

The molecular mechanism of dicarboxylic acid transport in Escherichia coli K 12.

It is the purpose of this communication to review the properties of the dicarboxylic acid transport system in Escherichia coli K 12, in particular the role of various dicarboxylate transport proteins, and the disposition of these components in the cytoplasmic membrane. The dicarboxylate transport system is an active process and is responsible for the uptake of succinate, fumarate, and malate. Membrane vesicles prepared from the EDTA, lysozyme, and osmotic shock treatment take up the dicarboxylic acids in the presence of an electron donor. Genetic analysis of various transport mutants indicates that there is only one dicarboxylic acid transport system present in Escherichia coli K 12, and that at least 3 genes, designated cbt, dct A, and dct B, are involved in this transport system. The products corresponding to the 3 genes are: a periplasmic binding protein (PBP) specified by cbt, and 2 membrane integral proteins, SBP 1 and SBP 2, specified by dct B and dct A, respectively. Components SBP 1 and SBP 2 appear to be exposed on both the inner and outer surfaces of the membrane, and lie in close proximity to each other. The substrate recognition sites of SBP 2 and SBP 1 are exposed on the outer and inner surfaces of the membrane respectively. The data presently available suggest that dicarboxylic acids may be translocated across the membrane via a transport channel. A tentative working model on the mechanism of translocation of dicarboxylic acids across the cell envelope by the periplasmic binding protein, and the 2 membrane carrier proteins is presented.     

Interaction of pentoxifylline with human erythrocytes. III. Comparison of fluidity change of erythrocyte membrane caused by S-adenosyl-L-methionine with that by pentoxifylline

The change in fluidity by adding pentoxifylline to erythrocyte membranes was compared with that caused by S-adenosyl-L-methionine (SAM) by the method of electron spin resonance (ESR) spectroscopy. When SAM or pentoxifylline was added externally to the erythrocyte suspension (outside), the fluidity of the membrane bilayer was increased after incubation at 37 degrees C. However, the fluidity change in the inner part of the bilayer was relatively small compared to that in its outer part. These fluidity changes were dependent on the incubation time and the temperature. When the erythrocyte suspension was preincubated overnight at 4 degrees C in the presence of drugs (inside), the fluidity of the inner part of the membrane changed significantly. Nevertheless, that of the outer part of the lipid bilayer was not affected. Such an asymmetric fluidity change in the lipid bilayer was not observed by the addition of other xanthine derivatives such as caffeine, theophylline and theobromine. S-Adenosyl-L-homocysteine suppressed and MgCl2 enhanced the increase of the membrane fluidity by SAM or pentoxifylline. Furthermore, the effects of SAM and pentoxifylline on erythrocyte deformability were determined by a filtering technique method. In increasing order the additive effects of SAM and pentoxifylline on the erythrocyte filterability were SAM (outside) less than pentoxifylline (inside) less than pentoxifylline (outside) less than SAM (inside). These results suggest that pentoxifylline also affects the membrane fluidity through the enzymatic methylation of phospholipids.     

Lipid nanotubes and microtubes: experimental evidence for unsymmetrical monolayer membrane formation from unsymmetrical bolaamphiphiles

Unsymmetrical bolaamphiphiles, omega- [N-beta-D-glucopyranosylcarbamoyl] alkanoic acids, with even-numbered oligomethylene chains (12, 14, 16, 18, and 20 carbons) self-assembled in water to form lipid nano- and microtubes. The tubular assemblies were separated by centrifugation and examined by transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy to study the molecular packing within the tubular membranes. The nanotubes encapsulated the staining reagent phosphotungstate, which revealed them to be hollow cylinders up to several hundred micrometers long with 30-43-nm outer diameters and 14-29-nm inner diameters. By comparing the membrane stacking periodicity obtained from powder X-ray diffraction analysis of the dehydrated tubes with the molecular packing within single crystals, we found that the nanotubes consist of an unsymmetrical monolayer lipid membrane (MLM) in which the molecules are packed in a parallel fashion. This suggests that the inner surface of the nanotubes is covered with carboxy headgroups and the outer surface with 1-glucosamide headgroups. The inner diameters of the lipid nanotubes could be controlled in the range 17.7-22.2 nm in steps of approximately 1.5 nm/two carbons by varying the oligomethylene spacer length. The microtubes had three types of molecular arrangements. The first type was a symmetrical MLM in which the molecules were packed in an antiparallel fashion. The other two types had unsymmetrical MLM stacking with head-to-head and head-to-tail motifs. Increasing the number of oligomethylene spacers stabilized the unsymmetrical MLM structure in both nano- and microtubes.     

Collagen-binding peptide interaction with retinal tissue surfaces

One of the current challenges in treating age-related macular degeneration (AMD) is the surface modification of the retinal Bruch membrane. In this study, the collagen fibers of the inner collagenous zone of the Bruch membrane were identified as type I and type III. Subsequently, the adsorption of a collagen-binding peptide onto the inner collagenous zone surface was investigated. The collagen-binding peptide was able to bind specifically to the collagen fibers while maintaining the biological activity of the N-terminus biotin tag. These results indicate that the collagen-binding peptide may be used as an anchor to immobilize bioactive molecules on the inner collagenous zone surface of the Bruch membrane.     

Preparation of insulin-loaded PLA/PLGA microcapsules by a novel membrane emulsification method and its release in vitro

Uniform-sized biodegradable PLA/PLGA microcapsules loading recombinant human insulin (rhI) were successfully prepared by combining a Shirasu Porous Glass (SPG) membrane emulsification technique and a double emulsion-evaporation method. An aqueous phase containing rhI was used as the inner water phase (w1), and PLA/PLGA and Arlacel 83 were dissolved in a mixture solvent of dichloromethane (DCM) and toluene, which was used as the oil phase (o). These two solutions were emulsified by a homogenizer to form a w1/o primary emulsion. The primary emulsion was permeated through the uniform pores of a SPG membrane into an outer water phase by the pressure of nitrogen gas to form the uniform w1/o/w2 droplets. The solid polymer microcapsules were obtained by simply evaporating solvent from droplets. Various factors of the preparation process influencing the drug encapsulation efficiency and the drug cumulative release were investigated systemically. The results indicated that the drug encapsulation efficiency and the cumulative release were affected by the PLA/PLGA ratio, NaCl concentration in outer water phase, the inner water phase volume, rhI-loading amount, pH-value in outer water phase and the size of microcapsules. By optimizing the preparation process, the drug encapsulation efficiency was high up to 91.82%. The unique advantage of preparing drug-loaded microcapsules by membrane emulsification technique is that the size of microcapsules can be controlled accurately, and thus the drug cumulative release profile can be adjusted just by changing the size of microcapsules. Moreover, much higher encapsulation efficiency can be obtained when compared with the conventional mechanical stirring method.     

An in vitro comparison of microdialysis relative recovery of Met- and Leu-enkephalin using cyclodextrins and antibodies as affinity agents

Cyclodextrins and antibodies have been used as affinity agents to improve relative recovery during microdialysis sampling. Two neuropeptides, methionine-enkephalin (ME) and leucine-enkephalin (LE), were chosen to compare the use of cyclodextrins and antibodies as possible affinity agents for improving their relative recovery across polycarbonate and polyethersulfone membranes during in vitro sampling. Cyclodextrins (CD) including β-CD, 2-hydroxypropyl-β-cyclodextrin (2HPβ-CD), and γ-CD gave improvements of relative recovery for both peptides of less than 2-fold as compared to controls. Comparisons of relative recovery between tyrosine–glycine–glycine, tyrosine, and phenylalanine using different cyclodextrins in the perfusion fluid were also obtained. Inclusion of an antibody against met-enkephalin in the microdialysis perfusion fluid resulted in relative recovery increases of up to 2.5-fold. These results show that using antibodies as affinity agents during microdialysis sampling may be more effective agents to improve the relative recovery of these opioid neuropeptides.     

Co-localization and interaction of organic anion transporter 1 with caveolin-2 in rat kidney

The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OAT's functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [(14)C]p-aminohippurate and [(3)H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1's function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.     

Energy transfer to analyse membrane-integrated mitoxantrone in BCRP-overexpressed cells

The binding and the diffusion of mitoxantrone (MTX) through the plasma membrane was performed by F?rster resonance energy transfer (FRET) from the membrane fluorescent donor (4Di-10ASP) to the co-localized acceptor MTX. The MTX addition to living 4Di-10ASP-tagged cells resulted in the rapid quenching of the probe emission (1s), revealing the MTX binding to the outer leaflet. Then, a slower quenching (about 90s) occurred which corresponded to the MTX flip-flop into the inner leaflet. Changes of MTX integration into the plasma membrane were described in BCRP-overexpressed cells (HCT-116R) treated with (i) the BCRP inhibitor fumitremorgin C (FTC), (ii) cyclosporin A (CSA) and (iii) benzyl alcohol (BA). Treatments with FTC or CSA showed 80% and 40% higher flip-flop of MTX from the outer to the inner leaflet of HCT-116R cells. The addition of BA clearly increased the MTX integration into both outer and inner leaflets. Confocal fluorescence microscopy displayed that FTC, CSA and BA enhanced MTX accumulation in HCT-116R. In conclusion, Fumitremorgin C and agents modulating MTX accumulation resulted in higher MTX integration in the resistant cell membrane and could disrupt the membrane cohesion. This energy transfer method appears well-adapted to describe the drug diffusion through the plasma membrane of living cells.