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Scientists studying the physiology of animals do not have the luxury of those studying humans when it comes down to sample collection. Humans, in general, can sit still while blood or saliva samples are taken, whereas animals can become stressed or might have to be held down or, in extreme cases, anaesthetised. For those studying intelligent animals like primates, saliva collection is a preferred alternative, as long as it yields the physiological answers that they are looking for. It is less invasive than blood sampling and is painless, and primates can be trained to accept sampling. This procedure has been adopted by Japanese researchers who have been investigating the salivary content of steroid hormones in chimpanzees. Nobuyuki Kutsukake from the Graduate University for Advanced Studies, Kanagawa, with co-workers from the University of Tokyo, Aska Pharma Medical, Kawasaki, and the Chimpanzee Sanctuary Uto, Kumamoto, wanted to use saliva to track changes in the levels of cortisol and testosterone. Saliva is known to be sensitive to acute changes in their concentrations and displays diurnal variations. It mirrors the behaviour of the plasma-free steroid fraction which represents steroids which are not bound to proteins and are deemed biologically active. The two steroids were measured by tandem LC/MS with electrospray ionisation. Samples were spiked with deuterium-labelled internal standards before extraction and analysis. Using selected reaction monitoring, the quantification limits of testosterone and cortisol were 5 and 10 pg, respectively. This is better sensitivity than conventional enzyme or radio immunoassays, which can also suffer from cross reactivity. Mass spectrometry also has the advantage that it quantifies only the target steroids, and it can do that simultaneously in one experiment. The team collected saliva from segments of cotton rope given to the chimps to chew on. This is becoming an established technique but there are some concerns over interference from endogenous steroids in the rope. To counter this, they introduced a preliminary washing procedure using hot water and hot aqueous ethanol which removed 96% of the testosterone-like steroids, leaving average values of 1.75 pg/cm. These were considered negligible compared with the salivary concentrations which are in the region of 15-30 pg/mL. The ropes were treated with a flavoured drinks mix to attract the chimps but this also did not affect the levels of steroids. The cortisol-like steroid levels in the rope were about 1.8 pg/mL, far lower than the salivary cortisol concentrations of about 1 ng/mL. The LC/MS technique was then used to compare the salivary steroid concentrations with those of free steroids in plasma. There were strong positive relationships between the salivary levels and the circulating steroids levels of both steroids. In addition, the diurnal variation in steroid levels was clear and well-defined, similar to that observed in humans. The amounts of steroids collected on the rope after being chewed by the chimps were far lower than those in the direct saliva samples. Nevertheless, there were strong relationships between the direct and rope samples. Taken together with the plasma-saliva correlation, rope sampling of saliva appears to be a reliable medium for studying steroid physiology. In another comparison, the testosterone concentrations in saliva were similar to those measured by radioimmunoassay but the latter technique was unable to detect the differences between the rope and direct saliva samples. This also illustrates the suitability of the salivary LC/MS procedure. Kutsukake ventured that the sample collection technique and the biological validity of measuring salivary steroid concentrations could be applied to other primates and non-primates living in captivity, free range, or in the wild, although there might be some problems reclaiming the rope from the wild animals. The ability to measure changes in salivary levels over time, without inflicting stress on the test animals, will permit the effects of certain behaviour or incidents to be evaluated. In the long term, the team hopes to be able to study the causal relationship between behaviour and steroid hormone levels.
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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Chimp with the flavoured rope used to collect saliva. |
