Although there are numereous variants of protocols of animal studies for metabonomic investigations, most studies follow a protocol simlar to the protocol shown in the figure. The study begins with an adaptation period of the animals. This acclimatization period is very important for the animals getting used to food, environment, other animals, stress and many other factors. During the adaptation period the metabolite profiles of the different animals become more similar and stable. The in-life phase of the study begins with the dosing of the animals. The duration of the study depends on the expected effects and the purpose of the study. During the complete study (not only during the in-life phase) urine is collected at regular intervals. Pre-dose urine samples just before dosing allow referencing animals to themselves and allow identifying outlying animals. Urine samples during the acclimatization allow monitoring the adaptation of animals. Several sampling time points during the in-life phase of studies allow monitoring time trajectories of the study, which is major advantage compared to toxicogenomics and histopathology. A sampling time point just after dosing allows the identification of drug metabolites (drug related compounds, DRC). These drug metabolites are usually excreted during the first hours after dosing. In the case of NMR measurements, the signals of DRCs can overlap with the signals of other endogenous metabolites. Therefore an identification of the signals belonging to drug metabolites is important. Serum and tissue is usually collected at the end of the study, as serum collection is a massive intervention for most animals. Most studies use data from histopathology for validation and integration of the metabonomics results.
For chronic studies the drug is administered several times and the studies usually take longer time. In order to reduce the number of animal studies it is of utmost importance to add serum and urine collections to existing routine animal studies. Thereby the sampling time points have to be adapted to the existing protocols, whereby it is important to prevent effects, which influence metabolic profiles (for example food restriction).
Time coures of a typical single dose animal study.