Small molecules, microdialysis

The microdialysis sampling process which allows the monitoring of small molecules in circulation within an animal, is an example. An artificial capillary is placed in the tissue region of interest, and a sample is coUected via dialysis. In the case of a laboratory animal such as a rat, a probe is placed in the jugular vein under anesthesia. Elow rates ate of the order of 1 p.L/min. 

In vivo microdialysis is based on the principle of dialysis, the process whereby concentration gradients drive the movement of small molecules and water through a semipermeable membrane. In vivo microdialysis involves the insertion of a small semipermeable membrane into a specific region of a living animal, such as the brain. The assembly that contains this semipermeable membrane is called a probe, which is composed of an inlet and an outlet compartment surrounded by a semipermeable membrane (see O Figure 9-1). Using a microinfusion pump set at a low flow rate (0.2-3 /rL/min), an aqueous solution known as the perfusate is pumped into the inlet compartment of the microdialysis probe. Ideally, the... 

Microdialysis probe. Enlarged view of the lower end shows a semipermeable membrane that allows small molecules to pass in both directions, but excludes large molecules. 

Dialysis is the process in which small molecules diffuse across a semipermeable membrane that has pore sizes large enough to pass small molecules but not large ones. A microdialysis probe has a semipermeable membrane attached to the shaft of a hypodermic needle, which can be inserted into an animal. Fluid is pumped through the probe from the inlet to the outlet. Small molecules from the animal diffuse into the probe and are rapidly transported to the outlet. Fluid exiting the probe (dialysate) can be analyzed by liquid chromatography. 

You can demonstrate the size of colloidal particles with a dialysis experiment in which two solutions are separated by a semipermeable membrane that has pores with diameters of 1—5 nm.3 Small molecules diffuse through these pores, but large molecules (such as proteins or colloids) cannot. (Collecting biological samples by microdialysis was discussed at the opening of Chapter 25.)... 

Figure 5 Microdialysis probe. Artificial cerebral spinal fluid is perfused through the inlet, and small molecules can diffuse across the membrane. Fluid is collected at the outlet and analyzed.

More success was obtained using on-line solid phase extraction before offline analysis by capillary electrophoresis with matrix-assisted laser-desorption ionization (MALDI) MS [18]. Because microdialysis is a method used for analysis of small molecules and peptides, MALDI is not used frequently with microdia lysis sampling. Another disadvantage is that MALDI cannot be used on-line with the separation because it is a vacuum ionization technique. However, if the pep-tide is large enough (0 1000 Da) MALDI can be useful. For the analysis of peptides in dialysate an appropriate separation is important before mass spectrometric detection. In a comparison with direct sampling of dialysate in MALDI, capillary electrophoresis provides the high efficiency separations necessary to resolve all... 

With in vivo microdialysis (Delgado et al., 1972 Ungerstedt, 1984), the extracellular fluid sample is ftirther qualified by a small sack or loop of semipermeable dialysis membrane (Fig. 51B). Artifidal CSF is slowly circulated into and out of the dialysis r on at flow rates of 1 -5 il/min, so that recovery of small molecules across the membrane from the extracellular space is effective. In essence the probe acts as an artificial blood vessel which may be sampled externally as a function of time during basal behavioral periods, drug administration (via the probe or elsewhere), external stimuli, etc. 

Janie, E. M., and Kissinger, P. T. (1993). Microdialysis and ultrafiltration sampling of small molecules and ions from in vivo dialysis fibers. AACC TDM/Toxicol. 14(7), 159. 

The microdialysis technique requires that a small-diameter (<300 pm) dialysis tube be stereotaxically implanted in a defined brain area. Perfusion of the dialysis tube with Ringer solutions enables diffusion of small molecules down their concentration gradient from the brain extracellular fluid and into the tube. The substances in the collected dialysates can be identified and measured by various analytical methods, either on-line or after storage (4). Extracellular transmitter levels may be measured either under basal conditions or after evoked release using, for example, high KCl concentrations in the perfusate. Because some classes of neurotransmitters, (such as the amino acids and catecholamines) are rapidly cleared from the extracellular space by specific reuptake mechanisms (5), it is often of interest to examine dialysate levels of these transmitters after perfusion with substances that will block these reuptake mechanisms. 

Therefore, most microdialysis membranes will allow the rapid passage of small molecules but usually stmggle for higher recoveries of larger molecules due to their smaller diffusivity. It is also obvious that the diffusion coefficient is directly proportional to the temperature. Empirically, the diffusion coefficient for small molecules increases 1-2 % per degree centigrade. Thus, it is crucial to cany out the entire microdialysis experiment at a constant... 

A microdialysis probe is used in biology to sample small molecules in fluids without contamination by large molecules, such as proteins. For example, a probe... 

Microdialysis probe. Small molecules pass through the semipermeable membrane, but large molecules cannot. [Courtesy R. T. Kennedy and Z. D. Sandlin, University of Michigan.]... 

The principle of dialysis is based on the free movement of small molecules through a semipermeable membrane (thickness 9-30 pm), whereas larger molecules (e.g., proteins) are not able to cross. Small molecules move through diffusion from a high-concentration zone to a low-concentration zone. Scaling down this process to small hollow fiber membranes is referred to as microdialysis. This sample preparation technique can be used to isolate compounds from tissue or other complex samples. In contrast to most other sample preparation techniques used for biological samples, only the unbound free fradion of the analyte is isolated in this method. 

A novel sampling method in Uving organisms is microdialysis (Fig. 10.10). A semipermeable tube, commonly made of a cellulose derivate, is introduced into the body. Small molecules can permeate the tube wall and are accepted by an acceptor solution which transports the material towards the analyser. A sophisticated technical solution offered by the BAS company (Fig. 10.10) is based on a concentric arrangement of two tubings, which also allows infusion of agents into the organism. 

The chromatogram and bar graph show results of a study of aspirin metabolism in a rat. Aspirin is converted into salicylic acid by enzymes in the bloodstream. To measure the conversion rate, aspirin was injected into a rat and dialysate from a microdialysis probe in a vein of the rat was monitored by liquid chromatography. If you simply withdrew blood for analysis, aspirin would continue to be metabolized by enzymes in the blood. Microdialysis separates the small aspirin molecule from large enzyme molecules. 

Clough GF, Boutsiouki P, Church MK, Michel CC. Effects of blood flow on the in vivo recovery of a small diffusible molecule by microdialysis in human skin. Journal of Pharmacology and Experimental Therapeutics 2002, 302, 681-686. 

The most practical and widely used method of continuously sampling interstitial fluid is microdialysis. In microdialysis, a buffer solution is pumped through a small semi-permeable tube that has been placed into living tissue. Molecules in the tissue interstitial fluid diffuse into the buffer and are pumped out and collected for measurement (Figure 12.1). Much of the pioneering work on the development of microdialysis was done in the 1970s [10]. Its basic principles have been reviewed [11]. 

Figure 12.1 Principles of microdialysis. Fluid circulates from inner tube to outer tube. The outer tube is semi-permeable. Molecules small enough to enter the pores are captured in the fluid and flow out of the probe for sampling. Reproduced from A.J. Rosenbloom et al., Mat. Sci. Forum. 457-460, pp. 1463-6. Copyright (2004), with permission from Trans Tech Publications...

Integration of a dialysis membrane or coupling to a microdialysis probe can help to exclude many of the larger molecules such as proteins that are typically responsible for fouling the sensor elements of microchip analysis systems. The nature of the analysis will dictate whether coupling to a flowthrough or separation-based sensor must be achieved and the kind of detection elements to be employed (optical, electrochemical, etc.). One of the issues with microchip devices is the very small... 

In order to conduct microdialysis experiments, several other components are required. Syringe pumps are often used to control the perfusate flow rate. The pump has to be able to deliver flow rates precisely in the microliter per minute range. Tubing is needed to connect between the probe and the pump which drives the perfusion flow and, in some cases, between the probe and a sample collector as well. The total dead volume of tubing should also be maintained as small as possible to have better time resolution. The perfusion fluid is a medium resembling the composition of extracellular fluid with minimal or zero concentration of the molecules of interest. Dial-ysate exiting from the outlet of the microdialysis probe is usually collected in a vial for later analysis. It is also possible to coimect the outlet directly to an analysis instrument without using a collector, which is usually preferred, if possible, for its convenience and usually faster analysis results. 

One last variant of membrane-based LLE will be mentioned briefly. Microdialysis corresponds to a miniaturized version of dialysis techniques routinely used to remove small ions and molecules from biological extracts. At this time it is clear that there are many obstacles to be overcome before routine use of this approach in trace quantitative analysis can he established (Torto 2001). An example of successful application of the method to a specialized biological problem is the determination of basal acetylchohne in micro-dialysate from the brain striatum of awake and freely moving rats, in which the miocrodialysis prohe was interfaced to HPLC with tandem mass spectrometric detection (Zhu 2000). 

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