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

当有机体进食时，它会消化食物中的生化成分，以生长新的组织或获得维持自身生存的能量。在这样做的过程中，来自饮食不同部分的主要元素，例如C和N，被结合到消费者有机体的组织中。这就产生了一句谚语“你吃什么，你就是什么”。30多年前，研究人员意识到，构成食物中营养物质的主要元素，即13C/12C和15N/14N的稳定同位素组成在不同的植物和动物之间存在差异，这是由于不同的生物合成和代谢途径导致不同的生化成分，或者食物链底部的碳和氮源的稳定同位素组成不同。上述现象为研究包括史前人类在内的化石生物的饮食提供了一种巧妙的新方法。考古遗址骨骼遗骸中保存的生物化学物质的稳定碳和氮同位素特征与潜在食物的稳定同位素值进行了比较，从而可以推断消费者的饮食。这门新学科的名字叫“古饮食重建”。这一新领域取得成功的一个主要原因是，人们已经发现，在数千年前的古代骨骼遗骸中，主要的结构骨骼蛋白--胶原蛋白被广泛保存。一个主要的观察结果是，生物体的N同位素值高于其饮食。这种现象被称为“营养水平效应”。因此，食草动物的N同位素值高于它们吃的植物的N同位素值，而食肉动物的N同位素值高于它吃的草食动物的N同位素值。因此，一个人的N同位素值将介于他或她所吃的动植物的值之间。值得注意的是，尽管这一效应已被用于数千篇论文报道的研究中，但组成主要身体蛋白质的氨基酸(AAs)中N营养丰富的潜在机制尚不清楚。我们研究的目的就是弥合这一知识鸿沟。直到最近，碳同位素还存在着同样的问题，但由于我们开发了一系列新的分析技术，使得能够高精度地记录单个化合物的碳同位素组成，这一知识空白已经开始填补。在结构蛋白胶原的情况下，这涉及将其分解为其组成的氨基酸，从中可以确定其C同位素组成，揭示胶原C同位素信号的来源。通过这种方法，我们揭示了C是如何从不同的膳食大量营养素中并入的，并确定了使用全胶原蛋白测量不会看到的膳食成分。这一新项目的目的是对构成结构蛋白质的氨基酸的氮同位素组成进行类似的分析。氮同位素分析在技术上是非常困难的；然而，我们最近发表了史前人类及其饮食的第一个系统的AA-N同位素测定。我们将应用这种方法，通过分析动物不同组织和饲料中蛋白质的AA成分，来提供对N营养效应的必要了解，这些蛋白质来自：(I)独特的受控猪饲养实验(猪被认为是人类的优秀动物模型)，其中动物以植物和动物蛋白为基础的饲料饲养；(Ii)组织培养实验，将首次解决细胞和整个生物体的影响；(Iii)从海洋食物网中采样的关键生物，这些生物特别有趣，因为它们最强烈地表达N营养效应，以及(Iiv)一系列考古人类和动物骨骼遗骸，这些遗骸来自本应消费不同数量的海洋和陆地食物的地区，以揭示人口水平的变化。

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.