Presentation on theme: "Improved On-line Measurement of Bone Collagen D/H as Forensic Environmental Indicator Abstract The organic hydrogen isotope ratio (deuterium/hydrogen or."— Presentation transcript:
Improved On-line Measurement of Bone Collagen D/H as Forensic Environmental Indicator Abstract The organic hydrogen isotope ratio (deuterium/hydrogen or D/H) in the tissues of terrestrial organisms is related to D/H values of precursor hydrogen in diet and water from precipitation. D/H in (fossil) bone collagen potentially characterizes the (paleo)environment of an animal. However, the original (paleo)environmental isotopic signal of organic hydrogen is largely limited to carbon-linked hydrogen (C-H). In contrast, organic hydrogen that is chemically linked to other elements (e.g., oxygen O-H and nitrogen N-H) is more loosely bound and exchanges with ambient water, even during storage and sample preparation. Further, the fraction of exchangeable hydrogen in total hydrogen can vary among samples. The uncertainty from exchangeable hydrogen can be reduced via equilibration with isotopically known water vapors and subsequent mass-balance calculations arriving at the D/H of non-exchangeable hydrogen in collagen. Labor-intensive methods for isotopic equilibration of exchangeable organic hydrogen with water vapor have been used for more than 15 years. Here we present data from steam-equilibrated samples using a more efficient continuous-flow (i.e., on-line) approach using a ThermoFinnigan TC/EA fitted with an autosampler. Collagens from bones of modern White tail deer ( Odocoileus virginianus ) and Southern mule deer ( O. hemionus ) across climate gradients in the USA were prepared for our preliminary study. Traditionally, TC/EA samples are wrapped tightly in non-permeable silver capsules that limit the access of steam to collagen. Our samples were loaded into individual micro-perforated silver TC/EA cups. Cups were crimped shut and looked like small shopping bags. The perforations at the bottom of each cup were small enough that collagen could not spill out of the cup, but steam and gas could freely pass into and out of the cups. Steam equilibration of an entire carousel (up to 49 cups) in an equilibration chamber occurred overnight with isotopically known steam at 115ºC, followed by drying with dry nitrogen, cooling, rapid transfer of the carousel to the TC/EA, and determination of D/H. We present preliminary results from collagens and discuss advantages of the new method. Introduction Hydrogen stable isotope ratios D/H (expressed as δD values) in precipitation across North America show strong directional trends. Atmospheric water is generally the ‘heaviest’ (i.e., D-enriched) in the southeast and gets ‘lighter’ as air masses travel northwest and lose moisture to precipitation (Bowen et al. 2005). Local variations in D/H of meteoric water due to altitude are superimposed (e.g., Hobson 1999). Tissues of living organisms carry isotopic signatures of their environment. Trophic isotope signals permeate through the food chain and provide a basis for characterizing and constraining the geographic origin of an individual (Meehan et al. 2001). The use of hydrogen isotopes in biomass faces the difficulty that some organic hydrogen atoms are weakly bonded in biomolecules, exchange with hydrogen from water, and thus may lose their original biogenic isotopic information. Exchangeable hydrogen is also called labile hydrogen and occupies chemically functional groups like –OH, -COOH, - NH 2, and some specific carbon-linked positions (Schimmelmann et al. 2006). On average about 10 to 15% of total organic hydrogen is exchangeable (Chamberlain et al. 1997). Exchangeable hydrogen can be chemically eliminated from a few organic substances, such as cellulose, through the process of ‘nitration’. Most chemically more complex biochemicals and bulk tissue require a different approach whereby exchangeable hydrogen is equilibrated, and thus isotopically controlled, with waters of known isotopic compositions. Such equilibrations were traditionally performed off-line in chambers with stationary water vapor (Wassenaar & Hobson 2000) or in dynamic flow-through conditions (Schimmelmann 1991). A remaining problem was the labor-intensive nature of equilibrating organic samples in individual quartz tubes. Here we present a more efficient equilibration approach. Materials and Methods Origin of bones and other samples Samples of White tail deer bone representing six individuals, two mule deer individuals, and one desert tortoise were chosen for this preliminary study. Samples were obtained from a range of locations that were separated geographically and by altitude and exhibit widely different meteoric water δD values. All individuals were considered to have their water obtained locally and the feeding habits were unambiguously assigned based on their taxonomic identities. Collagen preparation Katarina Topalov, Arndt Schimmelmann, David Polly, and Peter E. Sauer Department of Geological Sciences Indiana University, Bloomington, IN Acknowledgements We acknowledge support by the U.S. Department of Energy, Basic Energy Research Grant DEFG02- 00ER We thank Melanie Everett for identification of mammal bones, David Bish for assistance with XRD analysis, and the following hunters and scientists for providing White tail deer bones: Robert M. Zink from Minnesota, Robert E. Fowler from Texas, Jonathan Rupp from Indiana, Steve Rinker from Maine, and Candace McCaffery of the Florida Museum of Natural History. References cited Bowen G.J., L.I. Wassenaar, K.A. Hobson, Global application of stable hydrogen and oxygen isotopes to wildlife forensics. Oecologia 143, Bowen G.J., C.C. Miller, Isotopes in Precipitation Calculator - Google Earth Lastwww.waterisotopes.org visited April 4, Chamberlain C.P., J.D. Blum, R.T. Holmes, X. Feng, T.W. Sherry, G.R. Graves, The use of isotope tracers for identifying populations of migratory birds. Oecologia 109, Coplen T.B, New guidelines for reporting stable hydrogen, carbon, and oxygen isotope-ratio data. Geochimica et Cosmochimica Acta 60 (17), Cormie A.B., H.P. Schwartz, J. Gray, Determination of the hydrogen isotopic composition of bone collagen and correction for hydrogen exchange. Geochimica et Cosmochimica Acta 58, DeNiro M.J., S. Weiner, Chemical, enzymatic and spectroscopic characterization of “collagen” and other organic fractions from prehistoric bones. Geochimica et Cosmochimica Acta 52 (9), Hobson K.A., Tracing origins and migration of wildlife using stable isotopes: a review. Oecologia 120, 314–326. Hobson K.A., L.I. Wassenaar, Linking breeding and wintering grounds of Neotropical migrant songbirds using stable hydrogen isotopic analysis of feathers. Oecologia 109, 142–148. Meehan T.D., C.A. Lott, Z.D. Sharp, R.B. Smith, R.N. Rosenfield, A.C. Stewart, R.K. Murphy, Using hydrogen isotope geochemistry to estimate the natal latitudes of immature Cooper's hawks migrating through the Florida keys. Condor 103, 11–20. Ricker W.E., Linear regressions in fishery research. Journal of the Fishery Research Board of Canada 30, Schimmelmann A., Determination of the concentration and stable isotopic composition of nonexchangeable hydrogen in organic matter. Analytical Chemistry 63, Schimmelmann A., R.F. Miller, S.W. Leavitt, Hydrogen isotopic exchange and stable isotope ratios in cellulose, wood, chitin, and amino compounds. In: (P.K. Swart, K.C. Lohmann, J. McKenzie, S. Savin, Eds.), Climate Change in Continental Isotopic Records, American Geophysical Union, Geophysical Monograph 78, Schimmelmann A., J.-P. Boudou, M.D. Lewan, R.P. Wintsch, Experimental controls on D/H and 13 C/ 12 C ratios of kerogen, bitumen and oil during hydrous pyrolysis. Organic Geochemistry 32, 1009–1018. Schimmelmann A., M.J. DeNiro, Preparation of organic and water hydrogen for stable isotope analysis: effects due to reaction vessels and zinc reagent. Analytical Chemistry 65, Schimmelmann A., M.D. Lewan, R.P. Wintsch, D/H isotope ratios of kerogen, bitumen, oil, and water in hydrous pyrolysis of source rocks containing kerogen types I, II, IIS, and III. Geochimica et Cosmochimica Acta 63, 3751–3766. Schimmelmann A., A.L. Sessions, M. Mastalerz, Hydrogen isotopic (D/H) composition of organic matter during diagenesis and thermal maturation. Annual Review of Earth and Planetary Sciences 34, Wassenaar L.I, K.A. Hobson Improved method for determining the stable-hydrogen isotopic composition ( D) of complex organic materials of environmental interest. Environmental Science & Technology 34 (11), 2354–2360. Isotopic analysis In order to reproducibly measure meaningful δD values, two aliquots of each collagen sample were equilibrated in two different water vapors with δD values of either -136‰ or +1173‰ to control the isotopic composition of exchangeable hydrogen. The resulting isotopic difference between pairs of bulk δD values for each collagen was used to calculate the percentage H ex of exchangeable hydrogen in total hydrogen. All collagens are chemically similar and should have comparable H ex values, which serves as quality control. Finally, an isotopic mass-balance calculation yielded δD n values of non- exchangeable organic hydrogen in collagen (Schimmelmann et al. 1999, 2001). Isotopic composition of site-specific mean annual meteoric precipitation Each sample location was characterized by elevation, latitude, longtitude and the interpolated likely δD value of meteoric precipitation relative to VSMOW from Bowen & Miller’s Online Isotopes in Precipitation Calculator - Google Earth (www.waterisotopes.org; Figure 11). In addition, a water sample from a natural oasis in Joshua Tree National Park was directly measured isotopically.www.waterisotopes.org Figure 11. Hydrogen isotope ratio in precipitation (USA) with the locations of bone samples. Adapted from the Online Isotopes in Precipitation Calculator Google Earth (Bowen & Miller 2007, Results and Discussion X-radiographic powder diffraction (XRD) analysis of deer collagen produced no sharp mineral-based diffraction peaks, in contrast to bones of unprocessed chicken, modern elk and Jurassic Seismosaurus (Figure 12). This confirms that our acid digestion procedure quantitatively removed all biominerals from crushed bone. Figure 12. Comparison of collagen from acid-treated deer bone (black) with modern unprocessed chicken bone (green), modern unprocessed elk bone (red), and fossil Jurassic Seismosaurus bone (blue) (data from Dave Bish, Indiana University). The hydrogen exchangeability H ex in % of total organic hydrogen (Table 1) averaged 20.9% with an encouragingly small standard deviation of only 0.4%. This confirms that (i) all kerogens are chemically similar and (ii) water vapor accessed comparable hydrogen pools in all collagen samples. In other words, none of the perforated silver cups acted as a bottle neck causing incomplete equilibration. Further, (iii) back-equilibration with atmospheric moisture of previously equilibrated collagens during the rapid transfer of carousels into the TC/EA carousel chamber did not result in significant isotopic noise. Table 1. Collagen samples with their locations (latitude, longitude and elevation), of collagens after equilibration in ‘heavy’ (+1173‰) and ‘light’ (-136‰) water vapors, their relative isotopic difference ΔδD, hydrogen exchangeability H ex (exch.), the δD n value of non-exchangeable hydrogen in collagen based on an average fractionation effect of = 80‰ between water and exchangeable organic hydrogen, and local precipitation δD (δ 2 H) relative to VSMOW interpolated by the Online Isotopes in Precipitation Calculator - Google Earth (Bowen and Miller 2007). We compared our δD n values of non-exchangeable hydrogen in collagen to regional meteoric δD values from Bowen & Miller’s (2007) Isotopes in Precipitation Calculator in order to evaluate the D/H correlation between collagen and trophic hydrogen sources. The correlation is strong between most collagen samples and their associated meteoric waters (Figure 13) and confirms a trophic link between the δD of water and the δD n of collagen. δD n values of two of California’s collagen samples from the arid Joshua Tree National Park indicate evapotranspirative enrichment of deuterium relative to the δD value of local spring water from an oasis. The enrichment may occur in plants’ leaf waters and/or in the body fluid of animals and is expected in extremely dry environments (Cormie et al. 1994; Bowen et al. 2005). Figure 13. Collagen δD n in correlation with meteoric water δD. The functional linear regression was calculated according to Ricker (1973). δD n values of collagens from three different bones of a single White tail deer found dead in 2007 near Bloomington in Indiana show coherently and unexpectedly low levels of deuterium. Such D-depletion is not consistent with Bloomington’s meteoric water δD and suggests that the deer originated from Montana, Idaho, or Canada and migrated south or southeast into Indiana. Figure 1. Bones used in the study. Our collagen preparation procedure utilized elements from published procedures (DeNiro & Weiner 1988). Each bone was assigned an individual number and photographed (Figure 1). Fresh bone samples remained frozen until the preparation of collagen. First, large bones were cut into smaller bone segments using a hack saw. Bone segments were mechanically cleaned from soft tissue, debris, blood, dirt, and bone marrow using a knife. Central parts of leg bones, entire vertebrae, and a rib were then broken into smaller pieces and manually crushed using a mortar and Figure 2. Demineralization of ground bone in acid. Figure 3. Collagen –final dry product. pestle. Crushed bones were freeze- dried and stored in plastic bags. Aliquots of crushed bone samples weighing about 0.5 to 5 grams were Collagen was filtered and freeze-dried. Finally, lipids in dry collagen powder were extracted with dichloromethane, followed by air drying of the extracted collagens and storage in capped glass vials (Figure 3). In order to test the efficiency of our collagen extraction method, sample JTNP 1 was subjected to X-radiographic powder demineralized in Pyrex beakers with 1N hydrochloric acid for one week at room temperature with occasional stirring, followed by extensive washing with deionized water until the pH was about neutral (Figure 2). diffraction (XRD) to ascertain the absence of mineral contamination (Bruker D8, with Cu K radiation, solid-state detector, collecting data from 2 to 70 degrees with a step size of 0.02 degrees, counting for 2 s at each step). For determination of δD values of collagen, aliquots of collagen weighing ~1 mg were loaded into silver cups (Costech Analytical Technologies Inc.) that had their bottom micro- perforated to allow the passage of gas and steam without spilling collagen (Figure 4). The tops of loaded cups were crimped shut (Figure 5). Up to 40 crimped cups with collagen were loaded into an elemental analyzer (EA) carousel with 49 positions (Figure 6). Figure 4. Hand micro-perforated silver TC/EA cups. Figure 5. The left silver TC/EA cup has been crimped shut. Figure 6. Up to 49 silver TC/EA cups can be loaded into a carousel A few positions held international standard IAEA-CH-7 polyethylene foil in non- perforated silver cups with δD = ‰. The carousel was placed into an aluminum equilibrator enclosure with steam/gas inlet and outlet. Isotopically exchangeable organic hydrogen was equilibrated in water vapor overnight at 115 3 C. Quantitative removal of steam from the enclosure and drying of the equilibrated collagens was achieved by flushing with dry nitrogen gas for 4 hours at 115 C. Figure 8. Transfer of reference materials in silver cups, in dry nitrogen atmosphere. Figure 7. Glove dome purged with dry nitrogen. Following cooling under a continued flow of dry nitrogen, the closed equilibrator was transferred into a glove dome that was subsequently purged with dry nitrogen (Figure 7). The equilibrator was temporarily opened under dry nitrogen to add heat-sensitive hydrogen stable isotope reference The equilibrator was closed again (Figure 9), removed from the glove dome, and the nitrogen-purged carousel was quickly transferred into the TC/EA autosampler that serves as an on-line peripheral to a Delta Plus XP stable isotope mass-spectrometer (Thermo Finnigan (Figure 10). δD values of water samples were measured via manual dual-inlet mass-spectrometry of elemental hydrogen that was prepared from water by passage of water vapor over hot uranium metal or in contact with Indiana Zinc (Schimmelmann & DeNiro 1993). materials in non-perforated silver cups into remaining open positions in the carousel (Figure 8), for example coumarin δD = +82.3‰ and C 36 n -alkane with a very negative δD = ‰. (http://mypage.iu.edu/~aschimme/hc.html)http://mypage.iu.edu/~aschimme/hc.html All hydrogen isotopic data are reported in standard δD notation according to Coplen’s (1996) guidelines relative to VSMOW (0‰) and normalized to SLAP (-428‰), with mass- spectrometric precision ±3% for δD. Calculated H ex values carry a precision of ±0.5%. Figure 9. Closing of the equilibrator. Figure 10. Transfer of carousel into the TC/EA autosampler. Conclusions Simultaneous steam-equilibration of dozens of collagen and/or other organic samples in an EA carousel with subsequent on-line continuous-flow D/H measurements via TC/EA is far more efficient than previously available methods. Our method also allows for reduced sample sizes of 0.3 to 1mg, depending on the hydrogen content of organic substrates. of microperforated and crimped TC/EA silver cups allowed for steam and gases to access collagens during equilibration, without the risk of spilling and cross- contaminating collagen powders. The isotopic uncertainty from exchangeable hydrogen was reduced via equilibration with isotopically known water vapors and subsequent mass-balance calculations arriving at the δD n of non-exchangeable hydrogen in collagen. Future Steps Most collagens in our preliminary study confirm a strong isotopic relationship between hydrogen in collagen and in meteoric water. However, the δD n results for an Indiana White tail deer suggest that some deer may roam. Our future work will include species that do not migrate over long distances, for example rodents. Currently we are analyzing bones from California sea lions to evaluate the hypothesis that marine mammals incorporate D-enrichment into their biomass due to the relatively high abundance of deuterium in sea water.