Protocols

There are, of course, variations depending on the type of subjects to be investigated, and either exclusively urine or saliva samples can be collected. Typically, for adult subjects, after the collection of a predose sample of urine or saliva, they are asked to drink an accurately weighed mixture of the isotopes to give the required enrichment in body water. A small sample of the dose should be retained for isotope analysis. The dose bottle is then rinsed with a further amount of water («50 ml) and this is also drunk. Most investigators fast their subjects for at least 6 h and may restrict food and water intake during the time when the isotopes are equilibrating in body water. If a plateau method is used for the determination of dilution spaces, the requirement is to collect a sample after equilibration is complete but before turnover begins to reduce enrichment. This will usually require a series of three samples collected at successive hourly intervals between 4 and 8h. If urine samples are used, the first one should be discarded. A further two samples are collected two or three biological half-lives apart. In most adult cases, experiments will last 14 days; however, for both the timing of the plateau samples and the length of time of the study, it is advisable to establish specific times for the population under investigation. If dilution spaces are to be calculated from the intercept of isotope disappearance curves, postdose samples should begin to be collected on day 1 postdose and on subsequent days during the measurement period. Minimally, samples should be collected at the beginning and end of the measurement period (e.g., days 1, 2, 13, and 14). If a plateau method is used, samples are best collected in the presence of the investigation team, but when the intercept method is used subjects can be instructed to collect, label, and store their own samples. A few ml of urine, or saliva are sufficient for analyses, and should be collected and capped immediately to avoid evaporation and possible contamination. For long-term storage, samples should be stored frozen but may be refrigerated in the short term and need not be frozen for shipping.

Experience suggests that often it is the dose administration and sample collection that cause method failures. A good technique and high precision are needed for enrichment measurements but samples can always be reanalysed. Failures consequent on poor technique in subject-related procedures cannot be rectified and can be costly, especially if they are repeated through a whole investigation. New users of the methodology are advised to test all procedures in pilot work before full-scale application in a study.

Enrichment of samples is best calculated in terms of fraction of the dose given; that is,

where E is isotopic enrichment, d is a weight (g) of dose diluted in T (g) tap water, and D is the weight (g) of dose given. Subscripts S, P, D, and T refer to postdose sample, predose sample, diluted dose, and tap water, respectively. The reciprocal of plateau values is the isotope dilution space (ND or NO). The reciprocal of the value at the time zero intercept of a plot of its log value vs time provides alternative dilution space estimates. The slope is the rate constant (Kd or KO).

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