Establishing collagen quality criteria for sulphur isotope analysis of archaeological bone collagen

Sulphur isotope measurements of bone collagen from archaeological sites are beginning to be applied more often, yet there are no clear criteria to assess the quality of the collagen and therefore the validity of the sulphur isotope values. We provide elemental data from different methods (DNA sequences, amino acid sequences and mass spectrometric measurements) which are used to establish a reliable system of quality criteria for sulphur isotope analyses of bone collagen. The difference in the amount of sulphur from fish and mammalian collagen type I led to the suggestion to use different criteria to assess the in vivo character of the collagen between these two categories. For establishing quality ranges, the bone collagen of 140 modern animals were analysed. The amount of sulphur in fish and mammalian bone collagen is 0.63 ± 0.08% and 0.28 ± 0.07%, respectively. Based on these results we define for mammalian bone collagen an atomic C:S ratio of 600 ± 300 and an atomic N:S ratio of 200 ± 100, and for fish bone an atomic C:S ratio of 175 ± 50 and an atomic N:S ratio of 60 ± 20. These quality criteria were then applied to 305 specimens from different archaeological contexts.     

Experimental chemical degradation compared to natural diagenetic alteration of collagen: implications for collagen quality indicators for stable isotope analysis

Stable isotopic ratios from archaeological bone collagen are valid palaeodietary indicators, but depend on sufficiently well preserved molecules and several collagen quality criteria have to be fulfilled (mostly collagen wt%, C%; N%, C/N molar ratio). For a reassessment of these quality criteria, and a better understanding of the chemical degradation of bone collagen, experimentally degraded modern bones and 54 archaeological human bones were investigated. In the course of the experimental degradation, alterations of isotopic ratios were paralleled by altered collagen quality criteria. The contrary was found in the case of the archaeological specimens. This implies that the commonly used collagen quality criteria may be insufficient and do not guarantee that stable isotopic values of the gelatine extracts will still represent the original biological signal.     

Isotopic Comparison of Hair,Nail and Bone: Modern Analyses

This paper presents a comprison of the isotopic values of eight pairings of hair keratin and bone collagen and 12 pairings of hair keratin and nail keratin taken from living humans resident in the U.K., with the aim of examining whether modern human isotopic data can be directly compared to archaeological isotopic data.Results showed that bone collagen was enriched relative to hair keratin from the same individual by +1·4‰ in δ¹³C and +0·86‰ in δ¹⁵N, with some small degree of variability. Isotopic comparison of hair keratin and nail keratin from the same individual showed that there is no significant difference between hair and nail keratin in δ¹³C, but that nail keratin is on average +0·65‰ more enriched in δ¹⁵N than hair.Differences in amino acid composition between hair keratin and bone collagen may account for the carbon isotopic differences between the two proteins, and there is no significant overall carbon isotopic difference between hair and nail. However there are significant isotopic differences for nitrogen in the two pairings, that differences in amino acid composition and turnover times cannot explain. We suggest that these results indicate that constancy of isotopic values between tissues, even for similar proteins, cannot be taken for granted.     

IN VITRO DECOMPOSITION OF BONE COLLAGEN BY SOIL BACTERIA: THE IMPLICATIONS FOR STABLE ISOTOPE ANALYSIS IN ARCHAEOMETRY*

To understand biogenic collagen type I decomposition and to establish how diagenesis may bias archaeometric data, modern mammalian bone was inoculated with a selection of ubiquitous soil bacteria. The presence of exogenous microbial biomass in the inoculated specimens was then checked microscopically prior to collagen extraction. The experimentally degraded bone collagen showed altered amino acid compositions, attributable to the selective breakdown of certain amino acids by the bacteria. While both the bulk collagen extract and the single amino acids exhibited shifts to more negative δ¹³ C-values, enrichment was recorded for general δ¹³ N, and a depletion trend relative to unaltered collagen was observed for individual amino acid δ¹³ N. One explanation for the enrichment of the global δ¹⁵N-values is cleavage of peptide bonds, which leaves ¹⁵ N within the substrate, while the change of ¹³C is mostly due to the altered amino acid composition. On the other hand, possible repolymerization of cleavage products under experimental conditions may also be responsible for the depletion trend of individual amino acid δ¹³C- and δ¹⁵ N-values. This paper discusses the results as a basis for the development of a method for the reconstruction of the isotopic abundance of the original collagen, using the amino acid composition of the degraded product, the contribution of individual amino acids to its global δ-values and of isotope discriminations implied in the microbial decomposition.     

Archaeological collagen: Why worry about collagen diagenesis?

DNA appears to decay by random chain scission resulting in a predictable range of fragment lengths. Collagen decay has also been modelled in this same way, although it has become increasingly evident that collagen decay does not follow this same pattern. Radiocarbon and stable isotope analysis now use ultra-filtration to isolate large fragments (>30% of original polymer length) even in Pleistocene bone. How then does collagen decay? This study contrasts experimentally degraded samples with collagen extracted from forensic, archaeological and fossil bone. In experimentally degraded bone, values for amino acid and elemental (C:N) composition, bulk δ ¹³C, δ ¹⁵N, and aspartic acid racemisation (AAR) changed very little until 99% of the collagen was lost, suggesting that the collagen triple helix and polypeptide chains remained remarkably intact. This suggestion was demonstrated directly by examining the integrity of individual polypeptide chains using cyanogen bromide (CNBr) cleavage followed by SDS-PAGE electrophoresis. In ancient samples, AAR values remain remarkably stable and the pattern of CNBr-cleavage was only replaced with a smear of smaller polypeptides in the oldest (Pleistocene) bones investigated. Smearing may reflect both modification of the methionine resides (the sites of CNBr-cleavage) and/or partial hydrolysis of the collagen molecule. The findings reveal why it is not usually necessary to worry about collagen diagenesis; it is mostly intact. However, evidence of partial deterioration of the oldest bone samples suggests that alternative purification strategies may increase yields in some samples.     

Fish ’n chips: ZooMS peptide mass fingerprinting in a 96 well plate format to identify fish bone fragments

Fish are a large, highly diverse, and anthropologically important group of vertebrates. However, fish bones are underrepresented in the archaeological literature because they are less stable than those of other taxa and identification of bone to species is often difficult or impossible. We explore a new identification system, ZooMS (Zooarchaeology by Mass Spectrometry), which is based upon protein barcoding. As proteins can be cleaved enzymatically and analyzed by mass spectrometry in a repeatable way, protein barcoding is used widely in microbiological contexts for quick and inexpensive protein identification; mass spectra reflect the differences in protein sequence and can therefore be reproducibly linked to a particular protein or protein fragment. ZooMS uses peptide fingerprinting of bone collagen as a method for rapid identification of archaeological bone. This has involved the identification of masses related to peptides of known sequence. For mammals, sufficient sequence information is available for this approach but for groups, such the teleost fish, species are highly diverse and there are few available collagen sequences. Here we report a preliminary investigation into the identification of fish species by peptide mass fingerprinting that does not require sequence information. Collagen mass spectra are used to identify eight species of bony fishes (Osteichthyes) from four orders: Clupeiformes, Salmoniformes, Gadiformes, and Perciformes. The method is applied to both modern and archaeological fish remains and offers the capacity to identify traditionally unidentifiable fish fragments, thus increasing the Number of Identified Specimens (NISP) and providing invaluable information in specialized contexts.     

Isolation and Isotopic Analysis of Individual Amino Acids from Archaeological Bone Collagen: A New Method Using Rp‐hplc

This paper presents a new method for the isolation and isotopic analysis of some individual amino acids from proteins. The technique and its constituent steps are discussed; then isotopic analyses of amino acids from several samples of bone collagen from the Late Roman site of Poundbury, Dorset, UK are presented. The applications of the method are discussed, as well as some advantages of this technique relative to other methods. Although developed for use with archaeological bone collagen, the technique is equally applicable to other proteinaceous materials. The use of reversed‐phase HPLC avoids problems of isotopic fractionation inherent in using ion‐exchange HPLC. Amino acids are isolated preparatively, allowing both carbon and nitrogen isotopic values to be measured on a single sample using CF‐IRMS. Since amino acids are isotopically analysed in an underivatized form (unlike GC‐C‐IRMS), the method also presents the possibility of collecting the CO₂ generated during CF‐IRMS: this would allow the subsequent dating by ¹⁴C‐AMS of individual amino acids isolated from archaeological samples.     

古代皮革劣化机理的研究

为探究古代皮革的劣化机理,以现代皮革和人工老化皮革作为古代皮革的参考样品,采用热重-傅里叶变换红外光谱(TG-FTIR)、热裂解气相色谱-质谱(Py-GC/MS)联用技术对皮革的热降解过程及其分解产物进行分析。结果表明,古代皮革与现代皮革的热降解过程类似,可分为两个阶段,第一阶段以水的挥发为主,第二阶段以胶原蛋白的热分解为主,主要发生氨基酸的脱水、脱羧、脱氨、脱氢、侧链断裂等化学反应,分解产物有水、二氧化碳、氨气、二酮哌嗪类化合物、异氰酸、氢氰酸、吡咯、甲烷等。此外,古代皮革中胶原蛋白、单宁以及脂类物质相对百分含量较现代皮革大幅降低,表明这些物质的降解或流失导致了古代皮革劣化的发生。TG-FTIR和Py-GC/MS联用技术是研究古代皮革劣化机理的有力手段,可用于量化评估古代皮革劣化程度。     

Effects of Sodium Hydroxide Treatment and Ultrafiltration on the Removal of Humic Contaminants from Archaeological Bone

This study compared bone collagen extraction techniques that included treatment with sodium hydroxide and 30 kDa ultrafilters using a set of well‐preserved, humic‐contaminated archaeological marine mammal bones. Treatment with sodium hydroxide was effective at removing humic contaminants from archaeological bone, although yields were significantly decreased. Yields were also significantly decreased by ultrafiltration although this study produced no evidence that 30 kDa ultrafilters were effective at selectively removing humic contaminants from archaeological bone. The combination of sodium hydroxide treatment and ultrafiltration did not produce superior results to the treatment involving only sodium hydroxide. Archaeological samples exhibiting darker colouration indicative of humic contamination should be treated with sodium hydroxide to remove these contaminants. Copyright © 2017 John Wiley & Sons, Ltd.     

A novel and non-destructive approach for ZooMS analysis: ammonium bicarbonate buffer extraction

Bone collagen is found throughout most of the archaeological record. Under experimental conditions, collagen is apparently preserved as an intact molecule, with amino acid compositions and isotopic profiles only changing when almost all of the protein is lost. The ubiquity of collagen in archaeological bone has lead to the development of the use of collagen peptide mass fingerprints for the identification of bone fragments—Zooarchaeology by Mass Spectrometry (ZooMS). We report a novel, but a simple method for the partial extraction of collagen for ZooMS that uses ammonium bicarbonate buffer but avoids demineralisation. We compared conventional acid demineralisation with ammonium bicarbonate buffer extraction to test ZooMS in a range of modern and archaeological bone samples. The sensitivity of the current generation of mass spectrometers is high enough for the non-destructive buffer method to extract sufficient collagen for ZooMS. We envisage that a particular advantage of this method is that it leaves worked bone artefacts effectively undamaged post-treatment, suitable for subsequent analysis or museum storage or display. Furthermore, it may have potential as a screening tool to aid curators in the selection of material for more advanced molecular analysis—such as DNA sequencing.     

Comparing the survival of osteocalcin and mtDNA in archaeological bone from four European sites

The small mineral-binding bone protein, osteocalcin, has been applied in a number of studies on ancient bone due to predictions of its long-term stability. However, the intact protein has not been shown to survive in ancient bone devoid of DNA, which is a much more phylogenetically informative biomolecule. In this investigation, the survival of osteocalcin is directly compared to the amplification of mtDNA in a set of 34 archaeological samples from four sites throughout Europe. We also present unpublished osteocalcin sequences of seven mammalian species in addition to the 19 published sequences to highlight phylogenetic limitations of this protein. The results indicate that the intact osteocalcin molecule survives less in archaeological samples than mtDNA and is more subject to the temperature of the archaeological site. Amino acid analyses show the persistence of the dominant protein collagen in samples that failed both osteocalcin and mtDNA analyses. The implications these findings present for biomolecular species identification in archaeological and palaeontological material are that, although proteins do survive beyond ancient DNA, osteocalcin does not appear to be the most ideal target.     

Sulphur isotope evidence for freshwater fish consumption: a case study from the Danube Gorges,SE Europe

To explore the use of sulphur isotopes as an indicator of the consumption of freshwater fish, we undertook sulphur isotope analysis on bone collagen extracted from humans and animals from five archaeological sites from the Danube Gorges region dating from the Mesolithic to the middle Neolithic periods. The results show a difference in the sulphur isotope values between freshwater and terrestrial ecosystems of 8.7‰. To reconstruct human diets, bone collagen from 24 individuals was analysed for carbon, nitrogen and sulphur isotopic values. The nitrogen isotope ratios ranged from 10.3 to 16.5‰ and the carbon isotope ratios ranged from −20.8 to −18.3‰. Low nitrogen isotope values were found for individuals with low sulphur isotope ratios reflecting the low sulphur isotopic values of the terrestrial animals. The highest human nitrogen isotope values coincided with higher sulphur isotope ratios, which are related to the higher sulphur isotope values of the freshwater fish. Intermediate human sulphur isotopic ratios between these two extremes showed mixed diets of both terrestrial and freshwater resources.     

A Simple(R) Model to Predict the Source of Dietary Carbon in Individual Consumers

Quantitative individual human diet reconstruction using isotopic data and a Bayesian approach typically requires the inclusion of several model parameters, such as individual isotopic data, isotopic and macronutrient composition of food groups, diet‐to‐tissue isotopic offsets and dietary routing. In an archaeological context, sparse data may hamper a widespread application of such models. However, simpler models may be proposed to address specific archaeological questions. As a consequence of the intake of marine foods, individuals from the first century ad Roman site of Herculaneum showed well‐defined bone collagen radiocarbon age offsets from the expected terrestrial value. Taking as reference these radiocarbon offsets and using as model input stable isotope data (δ¹³C and δ¹⁵N), the performance of two Bayesian mixing model instances (routed and concentration‐dependent model versus non‐routed and concentration‐independent) was compared to predict the carbon contribution of marine foods to bone collagen. Predictions generated by both models were in good agreement with observed values. The model with higher complexity showed only a slightly better performance in terms of accuracy and precision. This demonstrates that under similar circumstances, a simple Bayesian approach can be applied to quantify the carbon contribution of marine foods to human bone collagen.     

Chicken and Egg: Testing the Carbon Isotopic Effects of Carnivory and Herbivory

In bone, the spacing between δ¹³C in collagen and bioapatite carbonate is greater in herbivores than carnivores, with implications for understanding animal dietary ecology from surviving hard tissues. Two explanations have been proposed: varying diet composition or differences in physiology between herbivores and carnivores. We measured the isotopic effects of carnivorous and herbivorous diets on a single species, to test the effect of diet composition alone. Protein δ¹³C and δ¹⁵N and carbonate δ¹³C were measured on egg and bone from hens on different diets. Herbivorous hens had a +14.3‰ spacing between egg albumen and shell δ¹³C, compared to +12.4‰ for omnivorous hens, and +11.5‰ for carnivorous hens. The bioapatite–collagen Δ¹³C spacing was measured as +6.2‰ for herbivorous hens, and calculated as +4.3‰ for omnivorous hens, and +3.4‰ for carnivorous hens—similar to observed mammalian herbivore and carnivore bioapatite–collagen Δ¹³C differences. We conclude that a shift in diet composition from herbivory to carnivory in a single species does alter the bioapatite–collagen carbon isotopic spacing. Our data strongly suggest that this results from differences in the Δ¹³C_{bioapatite–diet} spacing, and not that of Δ¹³C_{collagen–diet}.     

Preliminary evidence for medieval Polish diet from carbon and nitrogen stable isotopes

In this pilot study, stable carbon and nitrogen isotopes from bone collagen and apatite of skeletons from the 11th and 12th century cemetery in Giecz, Poland are interpreted. Isotope values from a small number of fish and animal bones from the same archaeological site are also examined. The goal of this research is to provide preliminary evidence of diet for a group of medieval Polish peasants, with particular emphasis on sex-based differences in diet. Results of isotope analyses suggest diet of this early medieval population was omnivorous and terrestrial-based. Fish bones sampled exhibit low δ¹³C ratios, and in half of the cases are significantly enriched in ¹⁵N, indicating they are freshwater species. Human bones do not reflect these signatures, suggesting freshwater fish were not a significant source of dietary protein at Giecz. The ¹³C_coll from some human bones is enriched beyond what might be expected from an exclusively C₃ diet. Associated mammal bones do not exhibit similarly elevated δ¹³C_coll ratios, suggesting enrichment among humans is not due to consumption of animals foddered on C₄ plants. Two possible sources of ¹³C_coll enrichment are marine fish in diet and direct consumption of a C₄ plant, such as millet. The δ¹³C values obtained from bone apatite of a small subset of humans suggest that millet contributes to ¹³C_coll enrichment, although at least three individuals may have also consumed small amounts of marine fish. Sex-based differences in δ¹⁵N ratios indicate that men consumed relatively more animal products (meat or dairy) than did women. There is also a correlation between δ¹³C_coll and δ¹⁵N values in skeletons of men that is absent in women. These carbon and nitrogen isotope data are the first reported for any Polish population and contribute to a more complete picture of dietary adaptation and social organization in medieval Europe.     

Polarizing Microscopy Identification of Chemical Diagenesis in Archaeological Cementum

Cementum increment analysis can potentially retrieve relatively complete, high-precision seasonality and mortality profiles from archaeological mammalian tooth assemblages. However, cementum exhibits many similarities to bone in composition, histology, ultrastructure, and even microstructure. Consequently, the mineralized dental tissue may be prone to the same processes of post-depositional chemical alteration that affect bone. This article reviews the issues surrounding chemical diagenesis in cementum and presents a new application to archaeological ungulate cementum of a polarizing microscopy technique that has previously been utilized to identify the effects of chemical diagenesis in archaeological bone and human cementum (Geusa et al., 1999; Gilbert, 1989; Watson, 1975; Zeder, 1978). It is argued that the post mortem leaching of collagen and the diagenetic growth of apatite crystallites can develop into banded features that mimic seasonal cementum increments. This pattern of diagenesis is demonstrated in multiple locations on a macroscopically well-preserved Upper Pleistocene-age archaeological tooth, which was previously interpreted by the author to exhibit biogenic seasonal cementum increments (Stutz, 1993; Stutz et al., 1995). It is suggested that if researchers seek to retrieve seasonality and mortality data from archaeological cementum, the microscopy analysis protocol must include screening of a pilot sample for the frequency and extent of chemical diagenesis.     

Stable isotopic composition of chitin from arthropods recovered in archaeological contexts as palaeoenvironmental indicators

The effects of biodegradation and heating on the stable carbon, nitrogen, hydrogen and oxygen isotope ratios of chitin in arthropods were studied. Chitinous exoskeletons from seven aquatic arthropod species were subjected to anaerobic marine biodegradation in mud, to terrestrial biodegradation in soils, and to thermal degradation under anaerobic and aerobic conditions. The isotope ratios of chromatographically separated D-glucosamine hydrochloride and derivatives from treated and untreated specimens were then compared. Carbon, nitrogen, oxygen and hydrogen stable isotope ratios were all found to be conserved during partial degradation of chitin. Micromorphological comparative studies using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated that no fungal chitin or other contaminants were present in the chitins that were chemically isolated from biodegraded substrates. Our results indicate that it will be possible to use stable isotope ratios of archaeological chitin samples for environmental and climatic reconstructions. An illustration of the utility of this approach comes from the observation that the stable isotope ratios of chitin from crustacean exoskeletons recovered from archaeological sites with ages up to 1400 years bp are in good agreement with measurements on modern crustaceans from similar environments.     

FOCUS: effect of diet and protein source on carbon stable isotope ratios in collagen: follow up to

We analyzed carbon stable isotope data from bone collagen of animals consuming varied experimental diets, including recently published data from Warinner and Tuross [Warinner, C., Tuross, N., 2009. Alkaline cooking and stable isotope tissue–diet spacing in swine: archaeological implications. Journal of Archaeological Science 36, 1690–1697; this journal]. Comparing regression lines for the relationship between collagen and diet δ¹³C, we show that protein source, and not physiology, explains the apparent taxonomic difference between swine and rodents reported in that paper. Our results reveal a complex relationship between whole diet and dietary protein in determining collagen δ¹³C values, such that in many cases, collagen alone may not provide reliable reconstructions of paleodiet. We advocate the simultaneous use of both collagen and apatite δ¹³C, whenever possible, to assess the diets of prehistoric peoples.     

Historical ecology of late Holocene sea otters (Enhydra lutris) from northern British Columbia: isotopic and zooarchaeological perspectives

We examined the isotopic composition (δ¹³C and δ¹⁵N) of sea otter (Enhydra lutris) bone collagen from ten late Holocene (ca. 5200 years BP–AD 1900) archaeological sites in northern British Columbia (BC), Canada. Because sea otters are now extinct from much of this region and have not fully recolonized their former range (e.g. Haida Gwaii and most of northern BC) these data represent an important first step towards better understanding sea otter foraging ecology in BC. The isotopic data suggest a diet composed primarily of benthic invertebrates, with a very low reliance on epibenthic fish. There is very low isotopic and thus inter-individual dietary variability in Holocene BC sea otters during the late Holocene. Furthermore, zooarchaeological abundance data suggest that otters represented a widespread and significant focus of aboriginal hunting practices on the northern BC coast during the mid- to late-Holocene. The consistent reliance on a small number of low-trophic level prey and limited dietary variability in Holocene BC sea otters may reflect top-down impacts on otter populations by aboriginal peoples. As part of our assessment of sea otter diet, we review trophic discrimination factors (Δ¹³C and Δ¹⁵N) for bone collagen from published literature and find marked variability, with mean values of +3.7 ± 1.6 for Δ¹³C (n = 21) and +3.6 ± 1.3 for Δ¹⁵N (n = 15).     

Stable isotopes in human skeletons of Southern Ontario: reconstructing Palaeodiet

Most native plants of Ontario use the C₃ photosynthetic mechanism and therefore have relatively low ${}^{13}\text{C}{}^{12}\text{C}$ ratios; maize is a C₄ plant and is relatively enriched in ¹³C. The proportion of maize in the diet of a human can therefore be estimated from the ¹³C-content of collagen of fossil bone. We have used this method to follow the increase in consumption of maize in ancient native populations over the interval from AD 400 to 1650, and to quantify the amount of maize consumed. Maize consumption rose gradually to a maximum of 50% of the diet by AD 1400. These data agree with archaeologically derived data for this region, and with previous isotope studies in the northeastern U.S.A. In some of the latter, however, the maximum percentage of maize consumed has been overestimated because maize was assumed to have a δ¹³C value of ?12·5‰; our studies of archaeological specimens show the correct value to be closer to ?9‰. The introduction of beans into the native diet about AD 1100, should have caused a decrease in the ¹⁵N content of human bone collagen because legumes are deficient in this isotope, with respect to meat and fish. However, we observe no significant change in the ${}^{15}\text{N}{}^{14}$N ratio of human bone collagen over the period from 2300 BC to AD 1640. We conclude that meat and fish remained the main sources of protein even after the advent of agriculture into this region.