Simultaneous Determination of Caffeine from Blood, Brain and Muscle Using Microdialysis in an Awake Rat and the Effect of Caffeine on Rat Activity

The purpose of this study was to demonstrate the ability to deliver a formulation and to monitor a drug in an awake, freely moving animal using simultaneous multiple-site microdialysis sampling. In addition, the movement of the rat was recorded before and after the dose so that the effect of the drug on rat activity can be correlated. In this study, caffeine was chosen as the drug for administration. Monitoring caffeine and its metabolites is widely performed in the study of induction of liver enzymes in drug interaction studies. Specifically, caffeine is used as the marker substrate for the cytochrome P450 isozyme designated 1A2, which metabolizes, among other things, tricyclic benzodiazepines such as clomipramine, clozapine and imipramine. Microdialysis sampling allows continuous, long-term sampling from the extracellular fluid of tissues with good temporal resolution and no net change in fluid volume in the animal. The protein-free samples can be analyzed directly on-line. The small size of the probes results in minimal perturbation to surrounding tissue, thus facilitating simultaneous sampling from several sites. In microdialysis sampling, an animal serves as its own control, thus reducing the number of animals needed for a given study, an advantage further enhanced by multiple-site sampling. Simultaneous multiple-site sampling facilitates continuous and long-term monitoring of parent compound and metabolite profiles from peripheral tissues and the bloodstream of the same animal. The BAS Raturn® accommodates numerous fluid lines and prevents their tangling, making it ideal for simultaneous multiple-site sampling. Movement by the animal triggers an optical sensor, activating the motorized turntable and signaling the software to record direction and duration of movement. The operator can record event markers for future reference. The methyl xanthines (i.e., theobromine, theophylline and caffeine), arewell-studiedcompounds,withmolecular weights of about 200 g/mol. Caffeine is a widely used—and perhaps abused—psychoactive drug. It has been shown to affect every body system that is controlled by the central nervous system (1). Caffeine has very low binding affinity to plasma proteins (2). It is metabolized in the liver by the CYP 1A enzymes system and is one of the compounds used for assessment of liver function (3). Because caffeine and its metabolites are well-characterized compounds and because caffeine could be expected to influence animal activity, we chose caffeine for this simultaneous multiple-site microdialysis sampling / activity monitoring study.