Application of in vivo microdialysis to measure leptin concentrations in adipose tissue

Microdialysis is a relatively new in vivo sampling technique, which allows repeated collecting of interstitial fluid and infusion of effector molecules into the tissue without influencing whole body function. The possibility of using microdialysis catheter with a large-pore size dialysis membrane (100 kDa) to measure concentrations of the adipocyte-derived peptide hormone leptin in interstitial fluid of adipose tissue was explored. Krebs–Henseleit buffer with 40 g/l dextran-70 was used to prevent perfusion fluid loss across the dialysis membrane. The relative recovery of leptin in vitro was determined using CMA/65 microdialysis catheter (100 kDa cut-off, membrane length 30 mm; CMA, Stockholm, Sweden) and four perfusion rates were tested (0.5, 1.0, 2.0, 5.0 μl/min). Furthermore, the microdialysis catheter CMA/65 was inserted into subcutaneous abdominal adipose tissue of 11 healthy human subjects and leptin concentrations in the interstitial fluid of adipose tissue in vivo were measured. The present findings are the first documentation on the use of microdialysis to study local leptin concentrations in the interstitial fluid of adipose tissue.     

Sensitive and specific method for the simultaneous determination of natural and synthetic catecholamines and 3,4-dihydroxyphenylglycol in microdialysis samples

The relatively new technique of microdialysis provides new possibilities for investigating in vivo the functioning of the sympathetic nervous system. The small sample volumes obtained, however, are a great challenge for analytical chemists. We report here a HPLC method for measuring in one run both natural and synthetic catecholamines [dopamine, (nor)epinephrine, -methylnorepinephrine, isoproterenol and epinine] and the intraneuronal metabolite 3,4-dihydroxyphenylglycol in small microdialysis samples after derivatization with the fluorogenic agent 1,2-diphenylethylenediamine. No prior clean-up step is necessary. N-Ethylmaleimide is necessary for preventing an inhibitory action on derivatization occurring in in vivo microdialysis samples. The method can handle large numbers of samples, is sensitive (on-column detection limits 30 to 200 fg) and reproducible (RSD 1 to 7%). Recovery characteristics of the commercial microdialysis probe used (CMA/20) were extensively investigated both in vitro and in vivo at various perfusion rates; for practical purposes a rate of 2 μl/min and sampling at 10-min intervals was found to be workable and to give good and reproducible recoveries (50 to 70%).     

Experimental and theoretical microdialysis studies of in situ metabolism

Microdialysis sampling was performed to monitor localized metabolism in vivo and in vitro. A mathematical model that accounts for analyte mass transport during microdialysis sampling was used to predict metabolite concentrations in the microdialysis probe during localized metabolism experiments. The model predicts that metabolite concentrations obtained in the microdialysis probe are a function of different experimental parameters including membrane length, perfusion fluid flow rate, and sample diffusive and kinetic properties. Different microdialysis experimental parameters including membrane length and perfusion fluid flow rate were varied to affect substrate extraction efficiency (E(d)), or loss to the sample matrix, in vivo and in vitro. Local hepatic metabolism was studied in vivo in male Sprague-Dawley rats by infusing acetaminophen through the microdialysis probe. Acetaminophen sulfate concentrations increased linearly with respect to acetaminophen E(d) in contrast to modeling predictions. Xanthine oxidase was used as an in vitro model of localized metabolism. In vitro experimental results partially matched modeling predictions for 10-mm probes. These results suggest that monitoring local metabolism using microdialysis sampling is feasible. It is important to consider system parameters such as dialysis flow rate, membrane length, and sample properties because these factors will affect analyte concentrations obtained during local metabolism experiments.     

Effects of Different Semipermeable Membranes on In Vitro and In Vivo Performance of Microdialysis Probes

The in vitro and in vivo performance of three different semipermeable microdialysis membranes was compared: a proprietary polycarbonate-ether membrane made by Carnegie Medecin; cuprophan, a regenerated cellulose membrane; and polyacrylonitrile. When microdialysis probes were tested in a stirred in vitro solution, large and statistically significant differences among the three membranes in extraction of acid metabolites (3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid, and homovanillic acid) and acetaminophen were found. Polyacrylonitrile had the highest extractions in vitro. In contrast, when microdialysis probes were implanted in vivo (in rat striatum), extraction of acid metabolites and acetaminophen did not differ significantly among the different membranes. These results are consistent with predictions made by a mathematical model of microdialysis and can be explained by the fact that in vitro the main factor limiting extraction is membrane resistance to diffusion, whereas tissue resistance to diffusion plays a more dominant role in vivo. These findings suggest that (aside from differences in surface area), the choice of semipermeable membrane will generally have little effect on in vivo microdialysis results. Furthermore, in vitro measurements of microdialysis probe extractions are not a reliable way of calibrating in vivo performance.     

A high recovery microsampling device based on a microdialysis probe for peptide sampling

A high recovery microsampling probe based on microdialysis was devised. The new probe showed a high recovery (100%) of peptides in vitro at different perfusion flow rates (0.1-1.0 μl/min). At a high flow rate, 1.0 μl/min, a 10-fold increased in recovery of peptides compared to the conventional microdialysis probe was achieved. A probe made of a low molecular weight cutoff membrane is suitable for filtering off proteins. The new probe can be a useful tool for high recovery of peptides from living tissues.     

Monitoring of met-enkephalin in vivo with 5-min temporal resolution using microdialysis sampling and capillary liquid chromatography with electrochemical detection

A method based on microdialysis sampling and capillary liquid chromatography with electrochemical detection that allows in vivo monitoring of met-enkephalin with 5-min temporal resolution is described. Sampling was achieved using a concentric microdialysis probe made from polycarbonate membrane material with a 20 kDa cut-off. This probe had an in vitro relative recovery for met-enkephalin of 63% at a dialysis flow-rate of 0.6 μl/min. Separations were performed using 7 cm×25 μm I.D. fused-silica capillary columns packed with 5 μm Alltima C₁₈ particles. A carbon fiber microelectrode was used as the detector electrode. The mass detection limit for met-enkephalin with this system was 40 amol. With on-column preconcentration, up to 2 μl of sample could be loaded onto the column resulting in concentration detection limits as low as 20 pM for met-enkephalin. Direct injection of dialysate, collected at 5-min intervals, allowed determination of met-enkephalin concentrations in the rat globus pallidus under basal and K⁺-induced depolarization conditions.     

Endogenous Release of γ-Aminobutyric Acid from the Medial Preoptic Area Measured by Microdialysis in the Anaesthetised Rat

The characteristics of gamma-aminobutyric acid (GABA) release as monitored by microdialysis have been investigated in the chloral hydrate anaesthetised rat. The high outflow of GABA following insertion of the microdialysis probe (membrane 2 mm in length, 0.5 mm in diameter) into the medial preoptic area was found to decline to a stable baseline level after 2 h. After this time, perfusion with a medium containing 100 mM potassium ions evoked a 56-fold increase in GABA outflow. The addition of the calcium channel blocker verapamil (100 microM) to the perfusion medium induced significant 25 and 50% reductions in basal and potassium-stimulated GABA outflow, respectively. In the same animals, verapamil caused an 80% decrease in potassium-stimulated noradrenaline outflow. The glutamic acid decarboxylase inhibitors 3-mercaptopropionic acid and L-allylglycine added to the perfusion medium at a concentration of 10 mM reduced basal GABA release by approximately 50% with different time-courses of action. Ethanolamine-O-sulfate, a GABA-transaminase inhibitor, induced significant increases in basal GABA outflow 90 min after inclusion in the perfusion medium. These results demonstrate that microdialysis is a suitable technique with which to monitor extracellular levels of GABA and provide in vivo data on GABA release and degradation mechanisms.     

A small, removable microdialysis probe

A miniaturized, concentric, microdialysis probe is described. It is constructed from 36 gauge stainless steel tubing inside of 26 gauge tubing, with a cellulose hollow fiber tip 0.2 mm in diameter and 2 mm long. It has a 6000 molecular weight cut off that excludes enzymes but collects monoamines, their metabolites, and other small neurochemicals. In vitro tests show relative recovery rates of 5-10%. Absolute recovery measured in picograms was independent of the perfusate flow rate inside the probe. Tests in awake rats with probes in the nucleus accumbens showed stable amounts of catecholamines and metabolites collected during repeated 20 min samples. After ip amphetamine, release of dopamine in the accumbens increased from 20 to 40 pg per sample while DOPAC and HVA decreased from about 1500 to 500 pg. Tests of multiple site sampling succeeded in obtaining norepinephrine and dopamine plus three metabolites (DOPAC, HVA and 5HIAA) from four probes simultaneously in four different brain sites in each rat. Five day continuous samples or monthly intermittent samples can be obtained with this microdialysis probe.     

High-performance liquid chromatographic analysis of muscular interstitial arginine and norepinephrine kinetics: A microdialysis study in rats

Complex interactions between the -arginine/nitric oxide synthase (NOS) pathway and the sympathetic nervous system have been reported. Methods capable of measuring -arginine and norepinephrine (NE) have mainly been reported for plasma. We report the use of the microdialysis technique combined with high-performance liquid chromatography (HPLC) for measurement of both -arginine and NE within the same tissue microdialysis sample. The microdialysis probe consisted of linear flexible probes (membrane length: 10 mm, outside diameter: 290 μm, molecular weight cut-off 50 kDa). The method used for -arginine measurement was HPLC with fluorescence detection, giving a within-run and a between-day coefficient of variation of 2.9 and 12.8%, respectively. The detection limit was 0.5 pM/20 μl injected for -/ -arginine. The method used for NE measurement was HPLC with electrochemical detection. The coefficients of variation were 4% for within-assay precision and 7.5% for between-assay precision. The detection limit for NE was 1 fmol/20 μl injected. The microdialysis technique coupled with HPLC system was validated in vivo to measure muscular interstitial concentrations of both arginine and NE under baseline conditions and after intravenous infusion of 500 mg/kg of -arginine or -arginine. In conclusion, the microdialysis technique coupled to HPLC allows the simultaneous measurements of both -arginine and NE within the same tissue microenvironment and will enable the study of the complex interactions between the -arginine/NO pathway and sympathetic nervous system within the interstitial space of different organs.     

Influence of Perfusate Glucose Concentration on Dialysate Lactate, Pyruvate, Aspartate, and Glutamate Levels Under Basal and Hypoxic Conditions: A Microdialysis Study in Rat Brain

Abstract: The aim of this study was to evaluate the influence of perfusion media with different glucose concentrations on dialysate levels of lactate, pyruvate, aspartate (Asp), and glutamate (Glu) under basal and hypoxic conditions in rat brain neocortex. Intracerebral microdialysis was performed with the rat under general anesthesia using bilateral probes (o.d. 0.3 mm; membrane length, 2 mm) perfused with artificial CSF containing 0.0 and 3.0 m M glucose, respectively. Basal dialysate levels were obtained 2 h after probe implantation in artificially ventilated animals. Dialysate levels of glucose were also measured for the two different perfusion fluids. The mean absolute extracellular concentration of glucose was estimated by a modification of the no-net-flux method to be 3.3 mmol/L, corresponding to an average in vivo recovery of 6% for glucose. Hypoxia was induced by lowering the inspired oxygen concentration to 3%. Hypoxia caused a disturbance of cortical electrical activity, evidenced by slower frequency and lower amplitudes on the electroencephalogram compared with prehypoxic conditions. This was associated with significant elevations of lactate, Asp, and Glu levels. There were no statistically significant differences in dialysate metabolite levels between the two perfusion fluids, during either normal or hypoxic conditions. We conclude that microdialysis with glucose-free perfusion fluid does not drain brain extracellular glucose in anesthetized rats to the extent that the dialysate lactate, pyruvate, Asp, and Glu levels during basal or hypoxic conditions are altered.     

Artificial feeding of northern fowl mites, Ornithonyssus sylviarum (Canestrini and Fanzago) (Acari: Macronyssidae), through membranes

A new device and technique are described for the in vitro feeding of northern fowl mites, Ornithonyssus sylviarum (Canestrini and Fanzago). The device consisted of a glass cylinder 25 mm in length and in outside diameter, capped with a chick skin membrane at one end and a snap-cap with a wire cloth window at the other end. Maximum feeding by northern fowl mites on warmed heparinized chicken blood occurred after 60 min and at a blood temperature range of 36-42 C. Skin membranes prepared from 1-wk-old chicks gave significantly higher feeding rates than those from 4-wk-old chicks, but unfrozen skins and skins frozen up to 4 wk were equally effective. Also, mites fed equally well through white leghorn and broiler chick skin membranes. About 80% of northern fowl mites fed. The in vitro technique described simplifies the approach to studies of northern fowl mite biology and physiology.     

Oxygen gradients in mitochondria examined with delayed luminescence from excited-state triplet probes

Phosphorescent probes are described that are quenchable by dioxygen and that partition into membranes. These probes are derivatives of porphyrin, in which the central metal, either zinc or palladium, induces intersystem crossing to enhance the triplet yield. The location of the probe in a suspension of membranes depends upon the charge distribution of side groups on the porphyrins. Probes that partition into the membrane are sensitive to phase transitions in lecithin artificial membranes. In the mitochondria these membrane probes are within transfer distance from tryptophans in membrane proteins. Although absolute concentrations of oxygen in membranes cannot be determined by this method, by comparing the oxygen dependence of a probe in the aqueous phase with that in the membrane phase under actively respiring and inhibited conditions, it is possible to examine the extent of O2 depletion at the mitochondrial surface. We show that at oxygen tensions of 0.2 microM and higher these gradients are insignificant at usual oxygen consumption rates of mitochondria.     

A steam-sterilizable electrode of the galvanic type for the measurement of dissolved oxygen

An oxygen probe is developed, using the principle of the silver-lead galvanic cell, covered with a plastic membrane. It is steam sterilizable while filled with water; after sterilization, water is easily exchanged for the electrolyte, potassium bicarbonate. Flat plastic membranes up to a thickness of about 0.1 mm can be applied. The membrane is secured and stretched in a special way, which guarantees a leakproof seal. The outer shape of the probe has a tulip-form and consists of polished stainless steel, except for the electrode face. The electrode is suitable for laboratory and industrial use and its components are completely standardized. Almost linear response for O₂-partial pressures up to atmospheric is obtained with 0.001 in. and 0.002 in. FEP-Teflon membranes. 90% response time is of the order of 10–15 sec for the 0.001 in. membrane, Residual current is almost negligible (< 1 μA).     

A novel method for the study of fluorescent probes in biological material during exposure to microwave radiation

Instrumentation has been developed which allows the monitoring of fluorescnece in erythrocyte ghost membranes before, during, and after exposure to microwave radiation. Using non-fluorescent, UV-transmitting transmitting fiber optic cables, excitation light of specific wavelengths was delivered to a stirred sample undergoing irradiation (2450 MHz, CW) within a fluid-filled, temperature-controlled waveguide. Fluorescence was collected using an identical cable and transferred through appropriate filters to standard detecting, amplification and recording devices. We have used the fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS)_to monitor the effect of mirowave radiation on the binding of calcium to erthrocyte ghosts. Microwave radiation at specific absorption rates of 10 and 200 mW/g had no effect on the binding of ANS to the membranes. Dose-responses curves also showed no influence of microwaves on calcium binding between 2.0 and 10.0 · 10^?4 M. In addition, experiments studying fluorescence energy transfer between intrinsic tryptophan residues and membrane bound ANS showed that intermolecular distance between donor and acceptor are also unaffected by microwave radiation. We have thus shown that 2450 MHz microwve radition at the specific absorption rates studies rates used does not interfere with the binding of calcium to erythrocyte ghosts or alter intermolecular distances intrinsic molecules and bound ANS.     

Aspects of neural plasticity in the central nervous system—III. Methodological studies on the microdialysis technique

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.     

Amino acid concentrations in hypothalamic and caudate nuclei during microwave-induced thermal stress: Analysis by microdialysis

Exposure to radiofrequency radiation (RFR) may produce thermal responses. Extracellular amino acid concentrations in the hypothalamus (Hyp) and caudate nucleus (CN) were measured by using in vivo microdialysis before and during exposure to RFR. Under urethane anesthetic, each rat was implanted stereotaxically with a nonmetallic microdialysis probe and temperature probe guides and then placed in the exposure chamber. The rat laid on its right side with its head and neck placed directly under the wave guide. Temperature probes were placed in the left brain, right brain, face (subcutaneously), left tympanum, and rectum. Each microdialysis sample was collected over a 20 min period. The microdialysis probe was perfused for 2 h before the rat was exposed to 5.02 GHz radiation (10 μs pulse width, 1000 pulses/s). The right and left sides of the brain were maintained at approximately 41.2 and 41.7 °C, respectively, throughout a 40 min exposure period. Initially when the brain was being heated to these temperatures, the time-averaged specific absorption rates (SARs) for the right and left sides of the brain were 29 and 40 W/kg, respectively. Concentrations of aspartic acid, glutamic acid, serine, glutamine, and glycine in dialysate were determined by using high-pressure liquid chromatography with electrochemical detection. In the Hyp and CN, the concentrations of aspartic acid, serine, and glycine increased significantly during RFR exposure (P < .05). These results indicate that RFR-induced thermal stress produces a general change in the amino acid concentrations that is not restricted to thermoregulatory centers. Changes in the concentrations of glutamic acid (Hyp, P = .16; CN, P = .34) and glutamine (Hyp, P = .13; CN, P = .10) were not statistically significant. Altered amino acid concentrations may reveal which brain regions are susceptible to damage in response to RFR-induced thermal stress. Bioelectromagnetics 18:277–283, 1997. © 1997 Wiley-Liss, Inc.     

Application of a new surface labeling reagent, EDTA derivative, on erythrocytes and platelets.

The modes of binding of a new class of impermeant metal-chelating probe, the complex of 111In3+ to 1-(p-benzenediazonium) ethylenediamine tetraacetic acid (azo-phenyl-EDTA), to human and rabbit erythrocyte membranes and the effect of binding on the function of rabbit platelets have been studied. The metal chelate, azo-phenyl-EDTA.[111In3+] bound covalently to membrane proteins following reaction with intact erythrocytes. The amount and the pattern of labeling was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide disc and slab gels for radioactivity. The pattern of labeling of intact human erythrocytes by azo-phenyl-EDTA.[111In3+], by pyridoxal phosphate-NaB3H7 and by galactose oxidase-NaB3H4 was also compared. The following results were obtained: (a) The pattern of labeling of intact human erythrocyte by azo-phenyl-EDTA.[111In3+] differed from other commonly used probes for labeling external membrane surfaces. Five polypeptides were labeled by the metal chelates. In addition to the known major proteins (protein band III, PAS-1, PAS-2 and PAS-3 of Fairbanks et al. (1972) Biochemistry 10, 2606--2617) a protein (radioactive band 4) which migrated slightly slower than PAS-3 in SDS gel was labeled heavily by the metal chelate. This protein has an apparent molecular weight of 37,500 in 8.4% acrylamide-SDS gel. About 40% of bound radioactivity was found in this protein. The diazo linkage of the metal chelate to this protein was found to be especially unstable to heat. (b) In rabbit erythrocyte membranes, the metal chelate bound to three polypeptides with apparent molecular weights of 96,000, 43,000 and 33,000 in 8.4% acrylamide gel. They are probably glycoproteins in nature. (c) The binding of the probe to platelets did not affect the platelet aggregability induced by adenosine diphoshpate. In vivo studies indicated that the labeled platelets accumulated at the plague of atherosclerotic rabbits. (d) The bifunctional analog of EDTA may permit new applications of metals with useful physical properties for studies of cell membranes.     

Chilling-induced changes in membrane fluidity and antioxidant enzyme activities in Coffea arabica L. roots

Exposure of coffee to low temperatures caused growth inhibition, changes in metabolic rates, and membrane alterations. Root tissue exposed to 10 °C evolved significantly lower rates of metabolic heat compared with controls grown at 25 °C, and the values were closely associated with the observed root growth inhibition. Electron paramagnetic resonance spectra of intact tissues showed that the spin probe 5-doxylstearic acid was capable to intercalate within the cellular membrane lipids. Indeed, at the depth of the 5th carbon atoms of the alkyl chains, the nitroxide radical detected more rigid membranes in seedlings exposed to 10 °C compared with 25 °C treated samples. Ascorbate peroxidase and catalase activities did not show appreciable changes under chilling conditions, while guaiacol peroxidase activity increased 55 % compared to the control. On the other hand, glutathione reductase activity decreased, in parallel to a significant decline in the capacity to reduce triphenyl-tetrazolium. Our results showed a marked correlation between lipid peroxidation and root tissue damage, which seemed to be associated with increased membrane rigidity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microdialysis for the determination of acetaldehyde and ethanol concentrations in the striatum of freely moving rats

The subject of the present paper is the simultaneous determination of ethanol (EtOH) and acetaldehyde (AcH) concentrations in the striatum of freely moving rats using an in vivo microdialysis followed by head-space gas chromatography (GC). Major operation conditions of GC were as follows: column, injector and detector temperatures 90, 110 and 200 degrees C, respectively; Supelcowax wide bore capillary column (60 m length, 0.53 mm i.d., 2 microm film thickness); carrier gas, nitrogen; flow rate, 20 ml/min. The recovery of EtOH and AcH at a concentration 40 mM and 250 microM, respectively, by microdialysis showed a maximum of 83.8+/-12.2 and 51.2+/-6.5%, respectively, at a flow rate of 0.8 microl/min. A good linear calibration curve in the concentration range of 5-50 mM for EtOH (r=0.998), and 10-250 microM for AcH (r=0.988) was observed. Microdialysates were collected for 10 min each after insertion of probe into the striatum. Rats were treated with cyanamide (100mg/kg, a potent aldehyde dehydrogenase inhibitor) and 60 min later with EtOH (1g/kg) intraperitoneally. A 10 min sample was about 8 microl. This volume was mixed with 40 microl of 0.002% t-butanol as an internal standard in 0.6N perchloric acid, and then analyzed by head-space GC method. The peak EtOH and AcH concentrations in the striatal dialysates reached maximum at 30 min, and then gradually decreased. This method represents a reasonable tool to quantify in vivo both AcH and EtOH levels simultaneously in rat brain.     

Agonistic interactions between Gambusia affinis and Galaxias maculatus: implications for whitebait fisheries in New Zealand rivers

Laboratory tank studies were used to identify the outcome of behavioural interactions between Gambusia affinis (gambusia) and Galaxias maculatus (inanga), whereas field data on spatial distribution were used to determine whether gambusia could be affecting inanga populations in the wild. Mortality rates for juvenile inanga (mean length 42 mm) exposed to gambusia (mean length 32 mm) for 15 days at 10°C were low (<10%) and no greater than those for control fish. However, at temperatures between 15 and 25°C, gambusia nipped the fins of juvenile inanga, resulting in immobilisation and death. Cumulative mortality rates over 15 days at these temperatures were over 60%. The survival of larger (mean length 58 mm) adult inanga was also reduced by gambusia. Adult inanga occurred in 15% of shallow (<0.5 m) sites where gambusia was present compared with 33% of shallow sites where it was absent. Furthermore, the mean depth of sites containing inanga in swamps and wetlands where agonistic interactions would be greatest was 1.8 m where gambusia was present compared with 1.0 m where it was absent. Displacement of inanga into deeper waters is therefore likely to be occurring as a consequence of gambusia aggression. The spread of Gambusia in New Zealand has the potential to reduce the use of shallow‐water habitats by inanga. This would further exacerbate the historic decline of these galaxiid fisheries related to land‐use changes.