Combined influence of meteoric water and protein intake on hydrogen isotope values in archaeological human bone collagen
Introduction
Stable isotope analysis of bones is relatively common in archaeology and paleontology to determine dietary components, provenance, migrations, climate proxies, metabolic functioning, and social demographics. Several decades of research have established a solid understanding of stable carbon, nitrogen, and oxygen isotope dynamics in archaeological bone collagen and hydroxyapatite. Hydrogen isotopes in bone have been addressed only recently. The routing of hydrogen into bone collagen in particular is less well-understood, but presents new options for understanding archaeological remains.
Hydrogen isotopes have been examined more thoroughly in tissues which are similar to the collagen protein and can serve as basic comparisons. Keratin (i.e. feathers, claws, nails) has been studied most heavily, although blood, muscle, lipids, and other organ tissues have been examined as well (Chesson et al. 2009, 2011; Hobson et al., 1999; Tuross et al., 2008; Wolf et al., 2011). Hydrogen is routed to keratin from both dietary food and drinking water, where the former pathway provides trophic information and the latter indicates latitudinal provenance (Bowen et al., 2005, 2009; Ehleringer et al., 2008; O'Brien and Wooller, 2007; Sellick et al., 2009). Where some studies suggest keratin hydrogen isotopes largely reflect drinking water isotope values (Hobson et al., 1999, Wolf et al., 2011), others suggest secondary dietary hydrogen input as well (Bowen et al., 2009; Ehleringer et al., 2008; Kirsanow and Tuross, 2011; Pietsch et al., 2011). Bulk blood, muscle, lipid, and organ hydrogen isotope values reflect largely drinking water sources (Chesson et al., 2011; Hobson et al., 1999; Wolf et al., 2011), although dietary input may have some influence (Commerford et al., 1983). While this previous research provides background for understanding hydrogen isotopes in bone collagen, keratin and other tissues have a more rapid turnover, a fundamentally different structure, and potentially different hydrogen sources rendering them inadequate proxies for collagen.
Collagen is the primary protein in animal bones and includes hydrogen atoms bound to carbon, or bound within carboxyl, amide, and minimal sulfhydryl side-groups. These side-group hydrogen atoms are labile and exchange with hydrogen from other water sources. The more stable carbon-bound hydrogen atoms comprise a calculated fraction of 0.742–0.829 (majority 0.77–0.81) of all hydrogen atoms in collagen (Cormie et al. 1994b, 1994c; Leyden et al., 2006; Sauer et al., 2009; Topalov et al., 2013) and are generally non-exchangeable with external water sources. The total (i.e. TOT) hydrogen isotope composition of bone collagen (i.e. COLL) can be represented asδ2HCOLL-TOT = (1-f) *δ2HCOLL-NEX + f *δ2HCOLL-EXwhere f represents fraction of exchangeable hydrogen (i.e. ∼0.19-0.23), δ2HCOLL-NEX represents the isotope value of non-exchangeable hydrogen atoms, and δ2HCOLL-EX represents the isotope value of exchangeable hydrogen atoms. Isotope values are in standard delta notation:δX = [(Rsample – Rstandard)/Rstandard]where R is the ratio (i.e. 2H/1H), values are in parts per thousand (‰), and the standard is V-SMOW.
The δ2HCOLL-NEX represents the isotope signal incorporated via water or dietary food and can be considered with a general conceptual framework:δ2HCOLL-NEX = (δ2Hingested water + εa) + (δ2Hdietary amino acids + εb).
The δ2Hingested water represents δ2H of water taken into the body via food water or direct drinking and incorporated into amino acids synthesized in vivo during collagen construction (i.e. “non-essential” amino acids). The εa represents hydrogen isotope fractionation during this process. The δ2Hdietary amino acids represents δ2H of amino acids synthesized ex vivo and are incorporated directly from consumed dietary proteins (i.e. “essential” amino acids). The εb represents subsequent fractionation as these amino acids are incorporated into collagen, although this value is suspected to be minimal and constant within a given species. Cormie et al. (1994a), Cormie et al. (1994c) and Chesson et al. (2011) present a thorough review of factors contributing to bone collagen δ2Hingested water, εa, δ2Hdietary amino acids, and εb; additional insight is gained from detailed discussions of keratin hydrogen incorporation (Ehleringer et al., 2008; Bowen et al., 2009).
A relatively strong linear correlation between δ2Hingested water and δ2HCOLL-NEX exists in strict herbivores obtaining all dietary fractions (i.e. amino acids, carbohydrates, water) from plants. Since leaf and stem δ2H values reflect local precipitation δ2H values, the herbivore δ2HCOLL-NEX correlates with these local precipitation δ2H values (Cormie et al. 1994a, 1994c; Pietsch et al., 2011; Reynard and Hedges, 2008). Hydrogen isotope values in herbivore bone collagen can be considered with the simpler representation of δ2HCOLL-NEX = δ2Hingested water + εa. Carnivores tend to show an apparent trophic level effect where δ2HCOLL-NEX deviates from the expected correlation with δ2Hingested water (Birchall et al., 2005; Pietsch et al., 2011; Reynard and Hedges, 2008; Topalov et al., 2013; Tuross et al., 2008). This is due likely to the additional δ2Hdietary amino acids variable which can show considerable range depending on the type and amount of animal protein consumed.
Humans present a complex case of omnivory. Limited research examining human collagen δ2H values suggests a combination of ingested water and dietary input (Reynard and Hedges, 2008), which agrees with limited data from other omnivorous mammals (Reynard and Hedges, 2008; Tuross et al., 2008). As archaeological human remains are of high interest, examining human collagen δ2H could provide another dimension by which to examine dietary input and ingestion of environmental water in a uniquely coupled pathway. It has the potential to contribute additional information to the study of geographic origin, migrations, and dietary choices or available foods.
This study uses the well-known relationships of bone nitrogen and oxygen with trophic structure and meteoric water, respectively, to explore these mechanisms’ effects on δ2HCOLL-NEX. Nitrogen in collagen (i.e. δ15NCOLL) is represented in standard delta notation as indicated previously where R is15N/14N and the standard is atmospheric air. The δ15NCOLL increases approximately 3–4‰ with trophic level (Bocherens and Drucker, 2003; DeNiro and Epstein, 1981; Schoeninger and DeNiro, 1984) providing a proxy for amount and type of dietary protein intake. Oxygen is found in the hydroxyapatite mineral fraction of bone in both the phosphate (i.e. PHOS) and carbonate (i.e. CARB) sites. Phosphate and carbonate oxygen isotopes (i.e. δ18OPHOS and δ18OCARB) are represented in standard delta notation where R is 18O/16O and the standard is V-SMOW. Both δ18OPHOS and δ18OCARB correlate with drinking water isotopes (Bryant and Froelich, 1995, Daux et al., 2008, Kohn, 1996, Longinelli, 1984, Luz and Kolodny, 1985, Luz et al., 1984) providing a proxy for geographic locality. The δ18O and δ2H values of meteoric water (i.e. MW) are strongly correlated according to the known meteoric water line: δ2HMW = 8 *δ18OMW +10 (Craig, 1961; Kendall and Coplen, 2001). In the absence of dietary influence, the δ2HCOLL-NEX is expected to correlate to δ18OPHOS and δ18OCARB with a similar slope to that of the meteoric water line. Deviations from this end member were compared to associated δ15NCOLL values, and multiple linear regression models constructed to determine the combined relative influence of ingested water and dietary proteins on the δ2HCOLL-NEX values. Combinations of δ2H and δ18O values in bone collagen have been used to examine herbivores, but this study adds to the sparser comparisons with δ15N, omnivores, and carnivores (Cormie et al., 1994a; Kirsanow and Tuross, 2011; Kirsanow et al., 2008; Pietsch et al., 2011; Topalov et al., 2013; Tuross et al., 2008).
Section snippets
Sample collection and preparation
Human remains were sampled from 11 North American archaeological sites primarily on the east coast with one southern site including individuals from Texas (Fig. 1, Table 1, Supplementary Table S1). These sites were selected based on availability of samples, range of geographic localities, and range of potential protein consumption. The sites are primarily temperate regions with similar humidity and temperature conditions. The exception is Glorieta Pass wherein the individuals hailed from the
Results
Samples adhering to the defined parameters for well-preserved collagen and hydroxyapatite were included in subsequent analyses. Table 3 includes all δ2H, δ18O, and δ15N data pertinent to statistical analyses and modeling. Table 4 includes full statistical results from regressions. Yield data, C:N ratios, FTIR data, and calculated differences between isotope values are presented in the Supplementary Tables S2 and S3. Although δ18OCARB is not used in any subsequent analyses or modeling, it is
Discussion
Hydrogen isotopes in human collagen present a complex combination of influences. Before discussing the influences of meteoric water and protein intake on the δ2HCOLL-NEX values, it is worth considering potential confounding factors in the oxygen and nitrogen proxy variables. These may include inter-regional movement of individuals, demographic factors, individual health status, variable isotope baselines of food, and non-local food sources.
Movement between regions and cities did occur in
Conclusions
Hydrogen isotopes in human bone collagen represent a combination of drinking water and dietary input. Using phosphate oxygen isotopes and collagen nitrogen isotopes as meteoric water and protein intake proxies, respectively, one can estimate the collagen hydrogen isotope values. This is contingent upon pooling multiple individuals from a given site to eliminate inherent variability from food choice, food availability, and movement between locations. Relationships between meteoric water oxygen
Acknowledgements
Collection access through Smithsonian National Museum of Natural History Skeletal Biology Program. K. Barca, K. Bruwelheide, C. Doney†, D. Dunn†, A. Lowe†, S. McGuire, S. Mills, W. Miller, J. Ososky, B. Pobiner, C. Potter, A. Warmack assisted with procurement/preparation of remains. Special thanks to D. Owsley (NMNH), and T. Coplen (USGS) for sample procurement and support. Thanks to T. Cleland, N. Sugiyama, and five anonymous reviewers for constructive comments.
†Supported by NSF REU Site Grant
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