Nitrogen isotopic discrimination in dietary amino acids: The threonine anomaly.

RATIONALE: The "Threonine Anomaly" relates to an observation made 25 years ago on the change in Thr nitrogen isotopic ratio in mammalian metabolism. Unlike all other amino acids, Thr in body protein is found to be depleted (rather than enriched) in 15 N relative to dietary Thr. Interpreting isotopic discrimination has become a useful source of ecological and palaeodietary information and it is desirable that the underlying processes are understood. METHODS: The principal enzyme of threonine catabolism, suggested to be responsible for the anomaly, threonine dehydratase, was prepared from rat liver. A time course of incubation of the enzyme with pure threonine was followed, and samples of residual threonine prepared for isotopic analysis by combustion in an automated carbon and nitrogen analyser coupled to a continuous flow isotope ratio mass spectrometer. RESULTS: We show experimentally, in vitro, that the enzymic reaction catabolising Thr cannot be responsible for its 15 N depletion. Plots of delta 15 N against both reaction time course and percentage completion show in fact an accelerating enrichment. CONCLUSIONS: A previously advanced suggestion that the unique catabolic mechanism for threonine was responsible for the anomalous depletion in 15 N is clearly not the case. We therefore offer alternative explanations, based on threonine's role at an organismal rather than cellular level. Copyright © 2016 John Wiley & Sons, Ltd.

Single amino acid radiocarbon dating of Upper Paleolithic modern humans.

Archaeological bones are usually dated by radiocarbon measurement of extracted collagen. However, low collagen content, contamination from the burial environment, or museum conservation work, such as addition of glues, preservatives, and fumigants to "protect" archaeological materials, have previously led to inaccurate dates. These inaccuracies in turn frustrate the development of archaeological chronologies and, in the Paleolithic, blur the dating of such key events as the dispersal of anatomically modern humans. Here we describe a method to date hydroxyproline found in collagen (~10% of collagen carbon) as a bone-specific biomarker that removes impurities, thereby improving dating accuracy and confidence. This method is applied to two important sites in Russia and allows us to report the earliest direct ages for the presence of anatomically modern humans on the Russian Plain. These dates contribute considerably to our understanding of the emergence of the Mid-Upper Paleolithic and the complex suite of burial behaviors that begin to appear during this period.

Community differentiation and kinship among Europe's first farmers.

Community differentiation is a fundamental topic of the social sciences, and its prehistoric origins in Europe are typically assumed to lie among the complex, densely populated societies that developed millennia after their Neolithic predecessors. Here we present the earliest, statistically significant evidence for such differentiation among the first farmers of Neolithic Europe. By using strontium isotopic data from more than 300 early Neolithic human skeletons, we find significantly less variance in geographic signatures among males than we find among females, and less variance among burials with ground stone adzes than burials without such adzes. From this, in context with other available evidence, we infer differential land use in early Neolithic central Europe within a patrilocal kinship system.

Evaluating marine diets through radiocarbon dating and stable isotope analysis of victims of the AD79 eruption of Vesuvius.

The stable carbon (δ(13) C) and nitrogen (δ(15) N) isotope values of bone collagen are frequently used in paleodietary studies to assess the marine contribution to an individual's diet. Surprisingly, the relationship between stable isotope these values characteristics and the percentage of marine foods in diet has never been effectively demonstrated. To clarify this relationship, the stable isotope values and radiocarbon dates of nine humans and one sheep from Herculaneum, all who perished simultaneously during the AD 79 eruption of Vesuvius, were determined. Significant differences were found in the radiocarbon dates which are attributable to the incorporation of "old" carbon from the marine reservoir. The magnitude of the observed differences was linearly correlated with both δ(13) C and δ(15) N values allowing the response of each isotope to increasing marine carbon in collagen to be independently verified. Regression analyses showed that for every 1‰ enrichment in δ(13) C and δ(15) N, 56 years and 34 years were added to the radiocarbon age, respectively. Predictions of the maximum marine reservoir age differed considerably depending on which stable isotope was considered. This discrepancy is attributed to some degree of macronutrient scrambling whereby nitrogen from marine protein is preferentially incorporated in collagen over marine carbon. It is suggested that the macronutrient scrambling explains the observed relationship between δ(13) C and δ(15) N from Roman coastal sites and should be considered when interpreting any diet which is not dominated by protein. Nevertheless, without knowing the degree of macronutrient scrambling in different dietary scenarios, the accuracy of dietary reconstructions is severely compromised.

Variation of bone collagen amino acid δ13C values in archaeological humans and fauna with different dietary regimes: developing frameworks of dietary discrimination.

We present bone collagen amino acid (AA) δ(13)C values for a range of archaeological samples representing four "benchmark" human diet groups (high marine protein consumers, high freshwater protein consumers, terrestrial C(3) consumers, and terrestrial C(4) consumers), a human population with an "unknown" diet, and ruminants. The aim is to establish an interpretive palaeodietary framework for bone collagen AA δ(13)C values, and to assess the extent to which AA δ(13)C values can provide additional dietary information to bulk collagen stable isotope analysis. Results are analyzed to determine the ability of those AAs for which we have a complete set, to discriminate between the diet groups. We show that very strong statistical discrimination is obtained for all interdiet group comparisons. This is often obvious from suitably chosen bivariate plots using δ(113)C values that have been normalized to compensate for interdiet group differences in bulk δ(13)C values. Bi-plots of non-normalized phenylalanine and valine δ(13)C values are useful for distinguishing aquatic diets (marine and freshwater) from terrestrial diets. Our interpretive framework uses multivariate statistics (e.g., discriminant analysis) to optimize the separation of the AA δ(13)C values of the "benchmark"' diet groups, and is capable of accurately assigning external samples to their expected diet groups. With a growing body of AA δ(13)C values, this method is likely to enhance palaeodietary research by allowing the "unknown" diets of populations under investigation to be statistically defined relative to the well-characterized or "known" diets of previously investigated populations.

Liquid chromatography/isotope ratio mass spectrometry measurement of δ13C of amino acids in plant proteins.

In archaeological studies, the isotopic enrichment values of carbon and nitrogen in bone collagen give a degree of information on dietary composition. The isotopic enrichments of individual amino acids from bone collagen and dietary protein have the potential to provide more precise information about the components of diet. A limited amount of work has been done on this, although the reliability of these studies is potentially limited by fractionation arising through hydrolysis of whole plant tissue (where reaction between amino acids and carbohydrates may occur) and, for certain amino acids, the use of derivatives (particularly trifluoroacetyl derivatives) for gas chromatography/isotope ratio mass spectrometry (GC/IRMS) analysis. The present study takes the approach of extracting the protein components of plant tissues before hydrolysis and using liquid chromatography/isotope ratio mass spectrometry (LC/IRMS), which does not require derivatisation, for measurement of the isotopic enrichment of the amino acids. The protocol developed offers a methodology for consistent measurement of the δ(13)C values of amino acids, allowing isotopic differences between the individual amino acids from different plant tissues to be identified. In particular, there are highly significant differences between leaf and seed protein amino acids (leaf minus grain) in the cases of threonine (-4.1‰), aspartic acid (+3.5‰) and serine (-3.2‰). In addition to its intended application in archaeology, the technique will be of value in the fields of plant sciences, nutrition and environmental food-web studies.

Using stable isotope analysis to examine the effect of economic change on breastfeeding practices in Spitalfields, London, UK.

Breastfeeding patterns were subject to a number of fads in 18th and 19th century Britain. Feeding infants by hand, rather than maternal breastfeeding or wet-nursing, became more prevalent among both the wealthy and poor. Substitute foods may have been a convenient alternative for mothers employed away from the household. This study used stable isotope ratio analysis to examine the weaning schedule in the 18th and 19th century skeletal assemblage from Spitalfields, London, UK. Analysis of 72 juvenile ribs revealed δ(15) N elevations of 2-3‰ above the adult mean for individuals up to the age of two, while elevations of 1-2‰ were observed in δ(13) C for the first year of life. This suggests that the introduction of solid foods took place before the end of the first year, and that breastfeeding had entirely ceased by 2 years of age. The age at death of many of these infants is known from historical records, and can be used to pinpoint the amount of time required for the breast milk signal to be observed in the stable isotope ratios of rib collagen. Results show that a δ(15) N elevation can be detected in the ribs of individuals who died as young as 5-6 weeks. Not all individuals at Spitalfields were breastfed, and there may not have been a single uniformly practiced weaning scheme. There is, however, more evidence for prolonged breastfeeding during the 19th century than the 18th century.

Amino acid delta13C analysis of hair proteins and bone collagen using liquid chromatography/isotope ratio mass spectrometry: paleodietary implications from intra-individual comparisons.

We report a novel method for the chromatographic separation and measurement of stable carbon isotope ratios (delta(13)C) of individual amino acids in hair proteins and bone collagen using the LC-IsoLink system, which interfaces liquid chromatography (LC) with isotope ratio mass spectrometry (IRMS). This paper provides baseline separation of 15 and 13 of the 18 amino acids in bone collagen and hair proteins, respectively. We also describe an approach to analysing small hair samples for compound-specific analysis of segmental hair sections. The LC/IRMS method is applied in a historical context by the delta(13)C analysis of hair proteins and bone collagen recovered from six individuals from Uummannaq in Greenland. The analysis of hair and bone amino acids from the same individual, compared for the first time in this study, is of importance in palaeodietary reconstruction. If hair proteins can be used as a proxy for bone collagen at the amino acid level, this validates compound-specific isotope studies using hair as a model for palaeodietary reconstruction. Our results suggest that a small offset observed in the bulk delta(13)C values of the hair and bone samples may be attributed to two factors: (i) amino acid compositional differences between hair and bone proteins, and (ii) differential turnover rates of the tissues and the amino acid pools contributing to their synthesis. This application proposes that hair may be a useful complementary or alternative source of compound-specific paleodietary information.

Radiocarbon and stable isotope investigations at the Central Rhineland sites of Gönnersdorf and Andernach-Martinsberg, Germany.

The late glacial open-air sites of Gönnersdorf and Andernach-Martinsberg in the German Central Rhineland are well known for their Magdalenian occupation and activities. The latter site also produced evidence for a younger, Final Palaeolithic occupation of the locality by people of the Federmessergruppen. Both sites are particularly well preserved, largely due to their burial beneath volcanic deposits of the late glacial Laacher See eruption. We conducted a program of AMS radiocarbon dating and stable isotope analyses with the aim of improving understanding of the chronological history and ecological setting of the two sites. Previously published radiocarbon dates appeared to indicate that the earliest Magdalenian occupation at Gönnersdorf fell around 12,900 uncalibrated (14)C yr BP, while the earliest occupation at Andernach may have been more than 500 radiocarbon years earlier. The AMS determinations presented here revise this impression and suggest that the onset of occupation at the two sites was in fact simultaneous and prior to the warming of Greenland Interstadial GI 1e. At Gönnersdorf, a chronological hiatus exists between the main Magdalenian faunal assemblage and mega-faunal remains interpreted as collected sub-fossil material. At Andernach-Martinsberg, there is a clear chronological hiatus between the Magdalenian occupation and subsequent Federmessergruppen activities at the site. However, an intermediate radiocarbon date on an atypically preserved horse bone is suggestive of ephemeral human visits to the site between these well demonstrated phases. A date of similar age on an elk bone from Gönnersdorf may indicate broadly contemporaneous human presence at Gönnersdorf too. Stable isotope analysis of faunal remains from Gönnersdorf and Andernach-Martinsberg was conducted with the aim of both reconstructing and comparing local environmental conditions at the two sites, and also potentially identifying subtle variations in the chronological development of the two sites not detectable at the level of precision of current radiocarbon dating techniques. No spatial trends in the faunal isotope signatures were observed within each site. In the case of samples with both radiocarbon and isotope data, no chronological pattern was observed for the isotope results. The Magdalenian faunal isotope signatures at the two sites resembled each other, suggesting comparable climatic and environmental conditions. The faunal delta(13)C signatures at Gönnersdorf and Andernach-Martinsberg were similar to those at contemporary European sites. While the faunal delta(15)N values were similar to those at contemporary sites in Germany, the UK, and Belgium, they were lower than those from the South of France. This difference in delta(15)N values is thought to relate to regional differences in the timing of changes in soil and plant nitrogen cycling in response to ameliorating climatic conditions.

Trans-Atlantic slavery: isotopic evidence for forced migration to Barbados.

The question of the ultimate origin of African slaves is one of the most perplexing in the history of trans-Atlantic slavery. Here we present the results of a small, preliminary isotopic study that was conducted in order to determine the geographical origin of 25 enslaved Africans who were buried at the Newton plantation, Barbados, sometime between the late 17th and early 19th century. In order to gain a more nuanced understanding of the slaves' origin, we used a combination of carbon, nitrogen, oxygen, and strontium isotope analyses. Carbon and nitrogen isotope ratios were determined in bone and dentinal collagen; oxygen and strontium isotopes were measured in tooth enamel. Results suggest that the majority of individuals were born on the island, if not the estate itself. Seven individuals, however, yielded enamel oxygen and strontium ratios that are inconsistent with a Barbadian origin, which strongly suggests that we are dealing with first-generation captives who were brought to the island with the slave trade. This idea is also supported by the fact that their carbon and nitrogen stable isotope values differ markedly between their teeth and bones. These intra-skeletal shifts reflect major dietary changes that probably coincided with their enslavement and forced migration to Barbados. While it is impossible to determine their exact origins, the results clearly demonstrate that the slaves did not all grow up in the same part of Africa. Instead, the data seem to suggest that they originated from at least three different areas, possibly including the Gold Coast and the Senegambia.

Metabolomics reveals diet-derived plant polyphenols accumulate in physiological bone.

Plant-derived secondary metabolites consumed in the diet, especially polyphenolic compounds, are known to have a range of positive health effects. They are present in circulation after ingestion and absorption and can be sequestered into cells within particular organs, but have rarely been investigated systematically in osteological tissues. However, a small number of polyphenols and similar molecules are known to bind to bone. For example alizarin, a plant derived anthraquinone and tetracycline (a naturally occurring antibiotic), are both absorbed into bone from circulation during bone formation and are used to monitor mineralization in osteological studies. Both molecules have also been identified serendipitously in archaeological human bones derived from natural sources in the diet. Whether an analogous mechanism of sequestration extends to additional diet-derived plant-polyphenols has not previously been systematically studied. We investigated whether a range of diet-derived polyphenol-like compounds bind to bone using untargeted metabolomics applied to the analysis of bone extracts from pigs fed an acorn-based diet. We analysed the diet which was rich in ellagitannins, extracts from the pig bones and surrounding tissue, post-mortem. We found direct evidence of multiple polyphenolic compounds in these extracts and matched them to the diet. We also showed that these compounds were present in the bone but not surrounding tissues. We also provide data showing that a range of polyphenolic compounds bind to hydroxyapatite in vitro. The evidence for polyphenol sequestration into physiological bone, and the range and specificity of polyphenols in human and animal diets, raises intriguing questions about potential effects on bone formation and bone health. Further studies are needed to determine the stability of the sequestered molecules post-mortem but there is also potential for (palaeo)dietary reconstruction and forensic applications.

Variability in the carbon isotope composition of individual amino acids in plant proteins from different sources: 1 Leaves.

The natural carbon isotope composition of individual amino acids from plant leaf proteins has been measured to establish potential sources of variability. The plant leaves studied, taken from a range of plant groups (forbs, trees, grasses, and freshwater aquatic plants), showed no significant influence of either season or environment (water and light availability) on their Δδ(13)C values. Plant groups did, however, differ in carbon isotope composition, although no consistent differences were identified at the species level. A discriminant analysis model was constructed which allowed leaves from (1) nettles, (2) Pooideae, (3) other Poales, (4) trees and (5) freshwater higher plants to be distinguished from each other on the basis of their natural abundance (13)C/(12)C ratios of individual amino acids. Differences in carbon isotope composition are known to be retained, to some extent, in the tissues of their consumers, and hence an understanding of compound-specific variation in (13)C/(12)C fractional abundance in plants has the potential to provide dietary insights of value in archaeological and ecological studies.

New hydroxyproline radiocarbon dates from Sungir, Russia, confirm early Mid Upper Palaeolithic burials in Eurasia.

Sungir (Russia) is a key Mid-Upper Palaeolithic site in Eurasia, containing several spectacular burials that disclose early evidence for complex burial rites in the form of a range of grave goods deposited along with the dead. Dating has been particularly challenging, with multiple radiocarbon dates ranging from 19,160±270 to 28,800±240 BP for burials that are believed to be closely similar in age. There are disparities in the radiocarbon dates of human bones, faunal remains and charcoal found on the floor of burials. Our approach has been to develop compound-specific methods using High Performance Liquid Chromatography (HPLC) to separate single amino acids, such as hydroxyproline, and thereby avoid the known human contamination on the bones themselves. Previously, we applied this technique to obtain radiocarbon dates of ∼30,000 BP for Sungir 2, Sungir 3 and a mammoth bone from the occupation levels of the site. The single amino acid radiocarbon dates were in good agreement with each other compared to all the dates previously reported, supporting their reliability. Here we report new hydroxyproline dates for two more human burials from the same site, Sungir 1 and Sungir 4. All five hydroxyproline dates reported are statistically indistinguishable and support an identical age for the group. The results suggest that compound-specific radiocarbon analysis should be considered seriously as the method of choice when precious archaeological remains are to be dated because they give a demonstrably contaminant-free radiocarbon age. The new ages are, together with the previously dated 'Red Lady of Paviland' human in the British Isles, the earliest for Mid Upper Palaeolithic burial behaviour in Eurasia, and point to the precocious appearance of this form of rite in Europe Russia.

Chronology of Ksar Akil (Lebanon) and implications for the colonization of Europe by anatomically modern humans.

The Out-of-Africa model holds that anatomically modern humans (AMH) evolved and dispersed from Africa into Asia, and later Europe. Palaeoanthropological evidence from the Near East assumes great importance, but AMH remains from the region are extremely scarce. 'Egbert', a now-lost AMH fossil from the key site of Ksar Akil (Lebanon) and 'Ethelruda', a recently re-discovered fragmentary maxilla from the same site, are two rare examples where human fossils are directly linked with early Upper Palaeolithic archaeological assemblages. Here we radiocarbon date the contexts from which Egbert and Ethelruda were recovered, as well as the levels above and below the findspots. In the absence of well-preserved organic materials, we primarily used marine shell beads, often regarded as indicative of behavioural modernity. Bayesian modelling allows for the construction of a chronostratigraphic framework for Ksar Akil, which supports several conclusions. The model-generated age estimates place Egbert between 40.8-39.2 ka cal BP (68.2% prob.) and Ethelruda between 42.4-41.7 ka cal BP (68.2% prob.). This indicates that Egbert is of an age comparable to that of the oldest directly-dated European AMH (Pestera cu Oase). Ethelruda is older, but on current estimates not older than the modern human teeth from Cavallo in Italy. The dating of the so-called "transitional" or Initial Upper Palaeolithic layers of the site may indicate that the passage from the Middle to Upper Palaeolithic at Ksar Akil, and possibly in the wider northern Levant, occurred later than previously estimated, casting some doubts on the assumed singular role of the region as a locus for human dispersals into Europe. Finally, tentative interpretations of the fossil's taxonomy, combined with the chronometric dating of Ethelruda's context, provides evidence that the transitional/IUP industries of Europe and the Levant, or at least some of them, may be the result of early modern human migration(s).

Correspondence between human diet, body composition and stable isotopic composition of hair and breath in Fijian villagers.

The main aim of this work was to describe the relationship between diet, and hair and breath isotopic composition. From one Fijian rural village, hair and breath samples were procured from 20 women. Physical anthropometrics were made, and hair (13)C/(12)C and (15)N/(14)N and breath (13)C/(12)C were measured. Individual diet diaries were kept for two of the four preceding weeks, and isotopic compositions of items which accounted for most of the diet were measured. Individual average diets were analysed for macronutrient and energy content and conform to reasonable nutritional expectation. Characteristics of the diet are described in terms of protein and energy, their patterning with respect to different clusters of food items and their relationship to individuals' anthropometry. Breath CO(2) is depleted in (13)C by 1-2 per thousand on average with respect to the total diet. Hair was enriched on average by 4.1 per thousand in nitrogen and 4.5 per thousand in carbon with respect to the total diet. There was insufficient population variation in hair isotopic composition to establish individual hair-diet isotopic differences. The definite relationship that we establish in this work, between dietary and tissue isotopic values for a human community, provides a basis for determining and validating dietary regimes more generally within non-industrial, non-global-'supermarket' economies.

Collagen turnover in the adult femoral mid-shaft: modeled from anthropogenic radiocarbon tracer measurements.

We have measured the (14)C content of human femoral mid-shaft collagen to determine the dynamics of adult collagen turnover, using the sudden doubling and subsequent slow relaxation of global atmospheric (14)C content due to nuclear bomb testing in the 1960s and 1970s as a tracer. (14)C measurements were made on bone collagen from 67 individuals of both sexes who died in Australia in 1990-1993, spanning a range of ages at death from 40 to 97, and these measurements were compared with values predicted by an age-dependent turnover model. We found that the dataset could constrain models of collagen turnover, with the following outcomes: 1) Collagen turnover rate of females decreases, on average, from 4%/yr to 3%/yr from 20 to 80 years. Male collagen turnover rates average 1.5-3%/yr over the same period. 2) For both sexes the collagen turnover rate during adolescent growth is much higher (5-15%/yr at age 10-15 years), with males having a significantly higher turnover rate than have females, by up to a factor of 2. 3) Much of the variation in residual bomb (14)C in a person's bone can be attributed to individual variation in turnover rate, but of no more than about 30% of the average values for adults. 4) Human femoral bone collagen isotopically reflects an individual's diet over a much longer period of time than 10 years, including a substantial portion of collagen synthesised during adolescence.

Analysis of amino acid 13C abundance from human and faunal bone collagen using liquid chromatography/isotope ratio mass spectrometry.

The scope of compound-specific stable isotope analysis has recently been increased with the development of the LC IsoLink which interfaces high-performance liquid chromatography (HPLC) and isotope ratio mass spectrometry (IRMS) to provide online LC/IRMS. This enables isotopic measurement of non-volatile compounds previously not amenable to compound-specific analysis or requiring substantial modification for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), which results in reduced precision. Amino acids are an example of such compounds. We present a new chromatographic method for the HPLC separation of underivatized amino acids using an acidic, aqueous mobile phase in conjunction with a mixed-mode stationary phase that can be interfaced with the LC IsoLink for compound-specific delta13C analysis. The method utilizes a reversed-phase Primesep-A column with embedded, ionizable, functional groups providing the capability for ion-exchange and hydrophobic interactions. Baseline separation of 15 amino acids and their carbon isotope values are reported with an average standard deviation of 0.18 per thousand (n = 6). In addition delta13C values of 18 amino acids are determined from modern protein and archaeological bone collagen hydrolysates, demonstrating the potential of this method for compound-specific applications in a number of fields including metabolic, ecological and palaeodietary studies.

Predicting diet, trophic level and palaeoecology from bone stable isotope analysis: a comparative study of five red deer populations.

C and N stable isotope ratios of red deer (Cervus elaphus) bone collagen (154 individuals) from five modern populations occupying geographically different habitats are reported. No significant difference was observed between deer occupying forested and non-forested environments subject to similar climatic conditions suggesting a simple "canopy effect" is not observed. Mean population delta13C is negatively correlated with temperature whereas mean population delta15N is positively correlated with temperature. A weak but significant positive correlation was observed between deer age and collagen delta13C values from the Isle of Rum population (Scotland). The amount of intra-population isotope variability is not consistent among populations; thus significant numbers of individuals from each species are required for modern food web studies, for palaeodietary baseline data, and for palaeoecological studies.

Preparative separation of underivatized amino acids for compound-specific stable isotope analysis and radiocarbon dating of hydrolyzed bone collagen.

Analysis of stable and radioactive isotopes from bone collagen provides useful information to archaeologists about the origin and age of bone artifacts. Isolation and analysis of single amino acids from the proteins can provide additional and more accurate information by removing contamination and separating a bulk isotope signal into its constituent parts. In this paper, we report a new method for the separation and isolation of underivatized amino acids from bone collagen, and their analysis by isotope ratio MS and accelerator MS. RP chromatography is used to separate the amino acids with nonpolar side chains, followed by an ion pair separation to isolate the remaining amino acids. The method produces single amino acids with little or no contamination from the separation process and allows for the measurement of accurate stable isotope ratios and pure samples for radiocarbon dating.