Unravelling the Nitrogen Isotope Signal of Skeletal Collagen for Palaeodeitary Reconstruction: The Origin of the Nitrogen Isotope Trophic Level Effect

When an organism eats it digests the biochemical components of the food to either grow new tissues or derive energy to sustain itself. In doing this the major elements, e.g. C and N, from the different parts of the diet are incorporated into the tissues of the consumer organism. This has given rise to the adage "you are what you eat". More than 30 years ago researchers realised that the stable isotopic compositions of the major elements making up the nutrients in the diet namely, 13C/12C and 15N/14N, vary between different plants and animals as a result of such factors as the differing biosynthetic and metabolic pathways giving rise to different biochemical components or the differing stable isotope compositions of the C and N sources at the base of food chains.

The above phenomena gave rise to an ingenious new way of studying the diets of fossil organisms, including prehistoric humans. Stable C and N isotope signatures of preserved biochemicals in skeletal remains from archaeological sites were compared with the stable isotope values of potential foods, allowing the diet of the consumer to be inferred. The new discipline has the title "palaeodietary reconstruction". A major reason for the success of this new field was the fact that the major structural skeletal protein, collagen, has been found to be widely preserved in ancient skeletal remains many 1000s of years old.

A major observation is that the N isotope value of an organism is higher than its diet. This phenomenon is termed the "trophic level effect". Thus, the N isotope value of herbivores are higher than that of the plants they eat, and the N isotope value of a carnivore is higher than that of the herbivores it eats. Hence, N isotope value of a human, will lie somewhere in between the values of the plants and animals he or she eats. Remarkably, although this effect has been used in research reported in several thousand publications, the underlying mechanics of N trophic enrichment within the amino acids (AAs) that comprise the major body proteins are unknown.

The aim of our research is to bridge this knowledge gap. Until recently the same problem existed for C isotopes but this gap in knowledge has begun to be filled as a result of us exploiting a range of new analytical techniques which allow the C isotope compositions of individual compounds to be recorded at high precision. In the case of the structural protein collagen this involves breaking it down to its constituent AAs, from which their C isotope compositions can be determined, revealing the origin of the collagen C isotope signal. Through this approach we have revealed how C is incorporated from different dietary macronutrients and identified dietary components which would not have been seen using whole collagen measurements.

The aim of this new project is to undertake analogous analyses of the N isotope compositions of the AAs that make up structural proteins. N isotope analyses are technically very difficult to perform; however, we recently published the first systematic determinations of AA N isotopes for prehistoric humans and their diets. We will apply this approach to provide the necessary understanding of the N tropic effect by analysing the AA components of the proteins in various tissues of animals and their diets, including those derived from: (i) a unique controlled pig feeding experiment (pigs being accepted as excellent animal models for humans) in which the animals were raised on both plant and animal protein based diets, (ii) tissue culture experiments which will allow cellular and whole organism effects to be resolved for the first time, (iii) key organisms sampled from marine food webs, which are particularly interesting as they express the N trophic effect most strongly, and (iiv) a range of archaeological human and animal skeletal remains from regions where varying amounts of marine and terrestrial foods would have been consumed, to reveal population level variation.

Our research addresses fundamental questions relating to the expression of stable isotope signals in human and animal tissues, making the primary end-users the palaeoecological and ecological communities. These researchers will benefit from our research via a better understanding of the fundamental controls on the stable isotope values, specifically of N in collagen amino acids (AAs), recorded in fossil tissues, which will undoubtedly inspire new research directions.

The methods we develop and the fundamental information we obtain is widely transferable to other disciplines in which protein metabolism and biochemistry are central, namely biomedicine, biogeochemistry, etc. Our analytical development work will expand the range of AAs for which isotope values can be routinely determined which will attract interest from the all communities performing such determinations. Compound-specific N isotope analyses at natural abundance are extremely challenging with only a handful of laboratories globally producing publishable values. This project will further consolidate and expand the availability of this technique to the UK NERC user community through the Bristol node of the NERC Life Sciences Mass Spectrometry Facility, which we host in our laboratories.

An unexpected impact of our pig feeding experiment emerged from discussions with our Co-I, Dr John Tarlton, who recognised that the animal tissues obtained will be of enormous value to researchers involved in the science of human aging. John will sample a wide range of tissues for examination of their biochemical and physiological properties. These investigations have the potential to afford crucial information influencing future interventions in combating the curse of osteoporosis, as well as directing studies into the prevention and cure of this debilitating disease.

We will engage with our primary scientific peers by publishing our work in the most prominent international peer reviewed journals and presenting at international conferences; it is for this reason that we have requested funds to present the outcomes of this work at 2 international conferences; the PIs also regularly give invited talks at other institutions and this work will be a focus of such presentations.

The general public have a seemingly insatiable interest in archaeology and we regularly promote interest in science via public and schools lectures in this area. With this is in mind at the start of 2009 we began the development of the 'The Palaeodetectives' display as an outreach tool to promote the science we undertake. We use cutting edge analytical chemistry to study of modern and ancient environments, on archaeological to geological timescales, and central to the display is a computer game played on touch-screens, which enables the user to learn about the natural world and how analytical chemistry is used to investigate it. The public are invited to 'solve cases' (previously published in high profile journals) from around the globe on the basis of historical, geographical and chemical information presented to them. The exhibit was originally created for the Royal Society Summer Science Exhibition 2009. As the feedback from this and other high profile events has been so praiseworthy we have decided to develop the game further and produce additional background information in order for it to be used by school teachers as an online resource in accordance with the National Curriculum guidelines. We regularly re-use the display at science festivals around the UK, and will offer it to our Swedish collaborators. We recognise that a limitation of public outreach activity is its potentially transitory nature; hence, we will construct a website that will be more long-lasting and serve as a link to other websites that discuss the role of stable isotope analysis in reconstructing ancient human diet. Our Impact Plan embeds our research into existing activities and provides new tools for dissemination.