Stable carbon and nitrogen isotope ratios of bone collagen: Variations within individuals,between sexes,and within populations raised on monotonous diets

The stable carbon and nitrogen isotope ratios of collagen of seven bones from each of three rabbits raised on a monotonous diet, and of two bones from each of eight female and seven male mink raised on another monotonous diet, were determined. The ranges of δ¹³C values and δ¹⁵N values were 0·5‰ and 0·6‰ for the rabbit bones and 1·0‰ and 1·4‰ for the mink bones. Uncertainties in the δ¹³C and δ¹⁵N values for prehistoric human diets estimated from the isotopic composition of collagen from the small numbers of bones which are typically available for analysis, and thus likely to be of the order of ±1‰.     

Pre-screening techniques for identification of samples suitable for radiocarbon dating of poorly preserved bones

Under certain environmental conditions, post-depositional diagenetic loss of bone collagen can severely reduce the number of bones from a particular archaeological site that are suitable for stable isotopic analysis or radiocarbon dating. This study examined nearly 300 bones from 12 archaeological sites across southern England known to yield poor or variable preservation to try to identify one, or more, pre-screening technique(s) that would indicate suitable collagen preservation for radiocarbon dating. The most reliable method was shown to be the percent nitrogen (%N) of whole bone powder, which has an 84% chance of successfully predicting whether or not a bone will yield sufficient (i.e. >1% weight) collagen for dating.     

Investigating the dietary life histories and mobility of children buried in St Gertrude Church cemetery,Riga, Latvia, 15th–17th centuries ad*

Carbon and nitrogen isotope profiles were obtained from incremental dentine analysis of 19 non-adults from a cemetery in Riga, Latvia. The research compared the life histories and diet between people buried in two mass graves and the general cemetery. The δ¹³C profiles of several children from the mass graves were similar but did not resemble the patterns seen in children from the general cemetery, suggesting that they probably represented a different population group. The rise in δ¹⁵N values towards the end of the life of four individuals from one mass grave suggests they were victims of an historically documented famine.     

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.     

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.     

The effect of growth on stable nitrogen isotope ratios in subadult bone collagen

Stable nitrogen isotopes have been used to reconstruct infant feeding practices as nursing infants have elevated δ¹⁵N ratios compared with their mothers. However, infancy is also a time of rapid growth, which may alter nitrogen isotope diet‐to‐tissue spacing. Several studies have documented a decrease in δ¹⁵N during growth in tissues with relatively fast accretion rates. This study investigates the effect that the growth of long bones, via collagen accretion, has on δ¹⁵N ratios. Long bones from individuals aged seven to nineteen years were obtained from a protohistoric ossuary in Ontario, Canada. Analysis of juveniles and adolescents permitted the examination of growth in a group who were not also nursing. It is concluded that a nitrogen isotope growth effect is not detectable in bone collagen from juveniles and adolescents, because: (1) δ¹⁵N ratios are not significantly different among the epiphyses, metaphyses and diaphysis of a growing long bone; (2) δ¹⁵N ratios are not significantly different between faster‐growing versus slower‐growing metaphyses; and (3) δ¹⁵N ratios are not significantly different between bones (or areas of a bone) that are still undergoing growth, versus bones that have ceased growing. The relatively slow speed of collagen accretion may explain why a growth effect is not manifested. Ultimately this research lends support to the use of nitrogen isotopes from bone collagen for infant feeding reconstructions. Copyright © 2009 John Wiley & Sons, Ltd.     

Investigation of the ‘canopy effect’ in the isotope ecology of temperate woodlands

Anomalously ¹³C-depleted carbon stable isotope values from closed forest ecosystems have been termed the ‘canopy effect’. Originally this was ascribed to recycling of depleted carbon from forest floor decomposition of organic material, although others have suggested that it is equally likely to be due to variations in leaf-level processes in response to increased shade. This depletion in the heavier carbon isotope is passed on to woodland herbivores feeding within the forest environments. A similar isotopic depletion has also been reported in the archaeological literature from more open temperate woodland settings, but no measurements have been made on the plants at the base of the food chain in order to quantify the effect. In this study we attempt this by examining the carbon and nitrogen stable isotopic values of different species of grasses from a range of open to closed habitat settings within Wytham Wood, Oxfordshire, UK. We find a strong relationship between carbon isotopic depletion of plant tissue and lowered light intensity with an up to 5‰ shift between grass grown in open and closed locations. In order to follow this up the food chain, we also report data on wool from sheep grazing in open pastures near the Wood, and on fallow deer living within the woodland, but which turn out not to show a strong canopy effect, probably related to their feeding strategies. We conclude that there is indeed a strong ‘canopy effect’ in temperate woodland, probably related to differential light levels, but that not all apparently woodland-dwelling mammals show such an effect. We also show considerable isotopic variation at the base of the food chain, which should counsel caution when attempting to interpret dietary isotopes using mixing models.     

Carbon isotope measurement techniques for bone collagen: Notes for the archaeologist

Stable carbon isotope analysis of archaeological human bone collagen is becoming increasingly useful in diet reconstructions. This paper describes, for archaeologists, the techniques that we have found useful in carrying out such analyses, and discusses the requirements for measurement accuracy and inter-laboratory calibration.     

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.     

Persistence of birefringence and specific collagen cross-links in dentine of teeth of the ‘Nuraghi’ population living in Sardinia BC 1500–1200

Well preserved bones of the Nuraghi population, discovered in 1980, are deposited in the Department of Biomedical Sciences, Division of Human Anatomy of the University of Sassari, in Sardinia, Italy. The Giant Tomb belongs to the megalithic monuments constructed in the period BC 1500–1200. The origin, history and the fate of Nuraghi population is not well known. Many cone-shaped fortresses are found in Sicily and megalithic monuments for burial purposes were built in their proximity. Due to the good macroscopic preservation of the mineralized parts of the Nuraghi skeletons we decided to compare the histological and biochemical structure of the recovered Nuraghi teeth with contemporary ones. Crown dentine of four Nuraghi molar and four premolar teeth were analysed and compared with contemporary molars and premolars removed for orthodontic reasons from 17–40-year-old patients in the Dental School, University of North Carolina at Chapel Hill USA. Evaluation of the collagen cross-links of dentine collagen was performed using a HPLC technique to analyze for hydrolysates. Nonreducible, stable cross-links pyridinoline and its lysyl analogue were quantified by fluorescence (excitation 330 nm, emission 390 nm). The concentration of reducible, labile cross-links referred to as DHLNL and HLNL (dehydro-dihydroxylisinorleucine) were identified and calculated by chromatography after the application of NaB³H₄ with integration of respective peaks and converting the numbers into a residue per mole of collagen basis. The ultrastructure of collagen was estimated by its persistent birefringence in polarized light after staining with Picrosirus Red. It was found that dentine collagen of Nuraghi teeth resisted environmental factors sufficiently well to preserve the molecular ultrastructure of collagen fibres, proved by their birefringence and by the preservation of some of the stable and reducible collagen cross-links, characteristic for skeletal tissues. © 1998 John Wiley & Sons, Ltd.