A Snapshot of the Active Chemistry in the Body by Microdialysis

Sara Bergström
Department of Analytical Chemistry
Uppsala University
March 2002

For certain drugs, like anesthetics, only a part of the drug dose administered help to relieve the pain. The drug binding to proteins, when the drug enters the blood stream, causes this effect. The bound part is not available for receptor interactions and can not create the pharmacological response. It is important to measure the active part, that is the non-bound fraction since the protein concentration and the drug binding are individual, to provide better dosing. As an analytical chemist, the two main points in my research are: determining active fractions of drugs in plasma and studying microdialysis - a minimal sampling unit that can be implanted in or used outside the body - as a tool in these measurements.

Primarily, microdialysis functioned as a tool for taking small samples from the brain. Today the working field for microdialysis has expanded and the technique is now also useful as an analytical tool. Microdialysis creates a snapshot of the chemistry in a certain body region, for example the stomach or a blood vessel, by collecting the molecules that for the moment appear in these regions. A special molecule then signalizes that a certain activity or process is going on in the body.

The heart of the technique is a cylindrical membrane -a material with channels. This membrane is about the same size as the tip of a pencil. A pump delivers a flow through the membrane and small molecules pass through the channels into this liquid, since nature struggles to reduce the differences in concentration between the solution inside and outside the membrane. This is called the equilibration step. The membrane excludes large molecules, like proteins. The selectivity in the sampling can be compared with the fisherman's net; only small fishes (or molecules) can pass the barrier. The out coming solution, the dialysate, is collected for analysis.

In some applications, researchers implant the small microdialysis unit in the body, for in vivo sampling. Other experiments apply the technique to body fluids outside the body - in vitro. In my research I determine active fractions of drugs in plasma, so far using in vitro experiments. The drug divides into a protein bound and a free fraction when it enters the blood stream and the problem is that only the free fraction creates the pharmacological response and makes the active fraction. My job is thus to determine the drug concentration in the active fraction. The protein binding varies for different drugs and it is especially important to measure the free, instead of the total concentration, when the binding is high. The protein concentration, and also the drug-protein binding, may vary between individuals and may be changed during for example surgery, certain diseases or trauma. This increases the significance of measuring the free and active concentration of the drug, in order to provide more specific and more individual drug dosing.

In a free fraction experiment, I place the membrane in a plasma sample and let the free drug molecules travel into the liquid inside the membrane. The proteins and the protein bound molecules are not allowed to pass and in that way I only collect the free concentrations of the drugs. The result is a snapshot of the active drug concentration.

Microdialysis is still a relatively new technique for free fraction extractions, but it is a good alternative. In my research, I work with a model substance (ropivacaine, a local anesthetic) with high degree of protein binding (about 90%) to evaluate microdialysis as an analytical tool in these measurements. So far the results have showed good agreement with the general used technique, ultrafiltration, where a filter retains the proteins. Microdialysis is advantageous due to the possibility of on-line coupling to the injection part of an analytical system. This simplifies the handling of the small sample volumes used.

One additional aim of my project is to use as small blood sample volumes from patients as possible. Today I use 0.2 ml (5 droplets) plasma samples, but ideally would be to perform the experiments in vivo - then the blood samples could be totally excluded. The trend in analytical chemistry is miniaturization of the techniques and with smaller instruments the "bed-side"-analysis, with the analytical apparatus next to the patient's bed, is perhaps not so far away. In this "bed-side"- analysis, microdialysis sampling should be an excellent tool.