For age calculation we use the Amino Acid Racemization Method. They are usually analysed using a gas-chromatograph (GC) or a high performance liquid-chromatograph (HPLC): Amino acid racemization dating
One of the greatest challenges confronting palaeontological, archaeological, palaeoenvironmental and palaeoclimatological studies, among others, is the need to place the observations on a chronostratigraphical scale. These reconstructions need to be achieved by a huge number of dating methods depending on the time scale, e.g. ESR, OSL, 210 Pb, 14 C or U/Th. Besides them, there is the amino acid racemization method, which can be applied to a large number of materials, including mollusk and ostracode shells. The amino acid racemization method is especially useful for the age range of 10 5 -10 6 yr B.P., which is, completely beyond the range of radiocarbon method and partially beyond that of U/Th method. Amino acid racemization has also been employed in dating Holocene materials, as well as younger samples from the last century or decades (e.g. Goodfriend, 1991; Goodfriend, 1992; Goodfriend et al. , 1992; Goodfriend et al ., 1995) and, even, in forensic determinations of age of death (Othani et al ., 1998).
The method basis relies on the fact that in most living beings amino acids are stereospecific, ...L-amino acids ( laevorotatory ). After the organism's death there is a progressive transformation of L-amino acids into D-amino acids ( dextrorotatory ) in a process called racemization, a reversible apparently first order chemical reaction that is temperature-dependent. When the D/L ratio approaches 1 the process is almost in equilibrium and the racemic state is attained, marking the method range. Nevertheless, amino acids with more than one chiral C atom at their molecule, as isoleucine, can undergo a process called epimerization, which consists on the transformation of the L-diastiomer (L-isoleucine) into a different D-diastiomer (D- Allo -isoleucine) not present in living beings. In this case the equilibrium is reached at a D- Allo /L-Ile ratio value of 1.3.
However, the amino acid racemization method is not a numerical dating method in isolation . There are two general approaches to calculate the age of a sample. The first one is based on the effects of time and temperature on the amino acid racemization/epimerization process, which may be determined in “high”-temperature laboratory experiments. These data together with the kinetic model equation can provide the age of a sample if its temperature history is known. The second approach consists on the calibration of D/L ratios with numerical datings in order to obtain age calculation equations.
Likewise, the racemization process is both genus and temperature dependent, so these algorithms can only be calculated from samples located in areas with the same thermal history.
Amino acid racemization provides some advantages over other dating methods. These can be summarised as follows:
1) Diverse materials, such as mollusc shells (Bada and Schroeder, 1972; Kriausakul and Mitterer, 1978; Wehmiller and Emerson, 1980; Briham, 1983; Kimber et al., 1986; Hearty et al., 1986; Goodfriend, 1987, 1989, 1991, 1992, among others), bones or teeth (Bada, 1972; Bada et al. , 1973; Belluomini, 1981; McMenamin et al. , 1982; Julg et al. , 1987; Torres et al. , 2000, 2002, among others), are suitable for this technique.
2) Only a small amount of sample, between 5 and 10 mg (or even less), is required for amino acid analysis by liquid-chromatography (HPLC) (Kaufman and Manley, 1998).
3) Many samples from a single bed can be analysed, thereby allowing anomalous results to be identified and time-averaging of the dated event to be calculated (Kowalewski, 1996, Kidwell, 1998; Kowalewski et al. , 1998; Behrensmeyer et al. , 2000; Kowalewski and Bambach, 2003).
4) This method has a greater range than other dating methods such as the radiocarbon technique. In the Iberian Peninsula the method range is ca. 1.3 Ma (Torres et al. , 1997; Ortiz et al. , 2004) but it also works very well for dating Holocene materials (e.g. Goodfriend, 1991; Goodfriend, 1992; Goodfriend et al. , 1992; Goodfriend et al ., 1995).
Ostracodes
Our experience (Ortiz et al ., 2000, 2002) indicates that ostracodes have significant characteristics that make them particularly useful for amino acid racemization/epimerization dating:
• Ostracode valves are mainly composed of low-magnesium-calcite (Sohn, 1958; Cadot and Kaesler, 1977; Bordegat, 1979, 1985) and initially racemize faster than gastopods (Ortiz et al ., 2002).
• In most cases, ostracodes are abundant and the only fossil fauna present in beds, so gastropods or bivalves cannot be used to obtain a complete and accurate amino acid chronology for a certain area.
• The excellent preservation of amino acids in ostracode valves means that only a small sample size (10-20 mg) is required for analysis by gas chromatography (GC), much less than for other organisms (e.g. molluscs 80 mg). Using reverse phase high performance liquid chromatography (HPLC), it is possible to analyze even a single ostracode valve ( cf . Kaufman, 2000).
• In a single GC analysis sample, there are typically between 1,500 and 2,000 ostracode valves, so the standard error or variance is low given the statistical significance of the sample size.
At the Biomolecular Stratigraphy Laboratory we are studying the Pleistocene paleoenvironmental evolution of different areas, where the ostracode species Cyprideis torosa (Jones) and Herpetocypris reptans (Baird) are very common.
| Age calculation algorithms for the D/L ratios of five amino acids (isoleucine, leucine, aspartic acid, phenylalanine and glutamic acid) analysed in continental ostracodes were determined for southern and central Iberian Peninsula and allow the numerical dating of deposits in the Mediterranean area since Lower Pleistocene time to present (Ortiz et al., 2004). |  Plots of first order kinetics transformed D/L ratios of ostracodes vs. time (for glutamic acid) and vs. square root of time (for aspartic acid). Asp: aspartic acid; Glu: glutamic acid. |
 |
For terrestrial gastropods from the central and southern Iberian Peninsula Torres et al. (1997) established the age calculation algorithms of different amino acids (leucine, isoleucine, aspartic acid, phenylalanine and glutamic acid) ranging from present to Lower Pleistocene (ca. 1.3 Ma) . |
Using ostracodes and gastropods we have established the:
• Chronostratigraphy of a 356 m-thick “composite-stratotype-section” of the east domain of the Guadix-Baza Basin (SE Spain), ranging from the Plio/Pleistocene boundary to the upper part of the Middle Pleistocene (Torres et al., 2004a).
• Chronostratigraphy of the Padul Basin (SE Spain), ranging from 1 Ma to 4.5 ka (Torres et al ., 2004b).
• Palaeontological sites in the Guadix-Baza Basin (Torres et al ., 1997; Ortiz et al ., 2000)
• Torralba and Ambrona palaeontological sites.
• Travertine fluvial terraces of Central Spain (Tagus and Ebro Basin) (Torres et al ., 1995, 2005; Ortiz et al ., 2009).
• Eolian deposits from the Canary Islands
• Archaeological sites (Covaciella, El Sidrón) (Fortea et al ., 1995, 2003).
References
Bada, J.L., 1972, The dating of fossil bones using the racemization of isoleucine, Earth and Planetary Science Letters, 15, 223-231.
Bada, J.L., Kvenvolden, K.A., Peterson. E., 1973, Racemization of amino acid in bones, Nature, 245, 308-310.
Bada, J.L., Schroeder, R.A., 1972, Racemization of isoleucine in calcareous marine sediments: kinetics and mechanism, Earth and Planetary Science Letters, 15, 1-11.
Behrensmeyer, A.K ., Kidwell, S.M ., Gastaldo, R.A ., 2000, Taphonomy and paleobiology, Paleobiology, 26, 103-147.
Belluomini, G., 1981, Direct Aspartic Acid racemization dating of human bones from archaeological sites of Central Southern Italy, Archaeometry, 23(2), 125-137.
Bordegat, A.M., 1979. Teneurs relative en phosphore, potassium et aluminium dans le caparace d´ostracodes actuels. Intérêt écologique (analyse à la microsonde électronique) In: Krstic, N. (Ed.), Taxonomy, Biostratigraphy and Distribution of Ostracodes. Proceedings of the 7th International Symposium on Ostracodes. Serbian Geological Society , Belgrade, Yugoslavia, pp: 261-264.
Bordegat, A.M., 1985. Composition chimique des carapaces d´ostracodes. Paramètres du milieu de vie. Atlas des ostracodes de France. Bulletin des Centres de Recherches et Exploration-Production Elf-Aquitaine 9, 379-386.
Brigham, J.K., 1983, Intrashell variations in amino acid concentrations and isoleucine epimerization ratios in fossil Hiatella arctica, Geology, 11, 509-513
Cadot, H.M., Kaesler, R.L., 1977. Magnesium content of calcite in caparaces of benthic marine Ostracoda. The University of Kansas Paleontological Contributions 87, 1-23.
Fortea, J., Rodríguez Otero, V. Hoyos Gómez, M., Valladas, H., Torres, T., 1995, Covaciella. Excavaciones arqueológicas en Asturias 1991-1994 , 4, 258-270
Fortea, J., Rasilla, M., Martínez, E., Sánchez-Moral, S., Cañaveras, J.C., Cuezva, S., Rosas, A., Soler, V., Castro, J., Torres, T., Ortiz, J.E. , Julià, R., Badal, E., Altuna, J., Alonso, J., 2003, La Cueva de El Sidrón (Borines, Piloña, Asturias). Campañas Arqueológicas de 2000 a 2002, Estudios Geológicos , 59 (1-4), 159-179.
Goodfriend, G.A., 1987, Chronostratigraphic studies of sediments in the Negev Desert, using amino acid epimerization analysis of land snail shells, Quaternary Research, 28, 374-392.
Goodfriend, G.A., 1989, Complementary use of amino-acid epimerization and radiocarbon analysis for dating of mixed-age fossil assemblages, Radiocarbon, 31, 1041-1047.
Goodfriend, G.A., 1991, Patterns of racemization and epimerisation of aminoacids in land snails shells over the course of the Holocene, Geochimica et Cosmochimica Acta , 55, 293-302.
Goodfriend, G.A., 1992, Rapid racemization of aspartic acid in mollusc shells and potential for dating over recent centuries, Nature , 357, 399-401.
Goodfriend, G.A., Hare, P.E., Druffel, E.R.M., 1992, Aspartic acid racemization and protein diagenesis in corals over the last 350 years, Geochimica et Cosmochimica Acta , 56, 3847-3850.
Goodfriend, G.A., Kashgarian, M., Harasewych M.G., 1995, Use of aspartic acid racemization and post-bomb 14 C to reconstruct growth rate and longevity of the deep-water slit shell Entemnotrochus adansonianus, Geochimica et Cosmochimica Acta , 59 (6), 1125-1129.
Hearty, P.J., Miller, G.H., Stearns, C.E., Szabo, B.J., 1986, Aminostratigraphy of Quaternary shorelines in the Mediterranean Basin, Geological Society of America Bulletin, 97, 850-858.
Julg A., Lafont R., Perinet G., 1987, Mechanisims of collagen racemization in fossil bones: application to absolute dating, Quaternary Sciences Reviews, 6, 25-28.
Kaufman, D.S., 2000, Amino acid racemization in ostracodes, in Perspectives in Amino Acid and Protein Geochemistry (eds. G. Goodfriend, M. Collins, M. Fogel, S. Macko, J. Wehmiller), 145-160, Oxford University Press, New York.
Kaufman, D.S., Manley, W.F., 1998, A new procedure for determining DL amino acid ratios in fossils using reverse phase liquid chromatography, Quaternary Geochronology, 17, 987-1000.
Kidwell, S.A., 1998, Time-averaging in the marine fossil record: overview of strategies and uncertainties, Geobios, 30, 977-995.
Kowalewski, M., 1996, Time-averaging, overcompleteness, and the geological record, Journal of Geology, 104, 317-326.
Kowalewski, M., Goodfriend, G.A., Flessa, K.W., 1998, High-resolution estimates of temporal mixing within shell beds: the evils and virtues of time-averaging, Paleobiology 24, 287-304.
Kowalewski, M., Bambach, R.K., 2003. The limits of paleontological resolution, in High resolution approaches in stratigraphic paleontology: topic in geobiology series 21 . (ed. P.J. Harries) , 1-48, Plenum Press / Kluwer , New York.
Kriasakul, N., Mitterer, R.M., 1978, Isoleucine epimerization in peptides and proteins: kinetics factorsand applications in fossil proteins, Science, 201, 1011-1014
McMenamin, M.A.S., Blunt, D.J., Kvenvolden, K.A., Miller, S.E., Marcus, L.F., Pardi, R.R., 1982, Amino acid geochemistry of fossil bones from the Rancho La Brea asphalt deposit, California, Quaternary Research, 18, 174-183.
Othani, S., Matsushia Y., Kobayashi Y., Kishi, K., 1998. Evaluation of aspartic acid in human femur for age estimation. Journal of Forensic Science 43, 949-953.
Ortiz, J.E., Torres, T., Llamas, J.F., Canoira, L., García-Alonso, P., García de la Morena, M.A., Lucini, M., 2000. Dataciones de algunos yacimientos paleontológicos de la cuenca de Guadix-Baza (sector de Cúllar-Baza, Granada, España) y primera estimación de edad de la apertura de la cuenca mediante el método de racemización de aminoácidos. Geogaceta 28, 109-112.
Ortiz, J.E., Torres, T., Llamas, F.J., 2002. Cross-calibration of the racemization rates of leucine and phenylalanine and epimerization rates of isoleucine between ostracodes and gastropods over the Pleistocene in southern Spain. Organic Geochemistry 33, 691-699.
Ortiz, J.E., Torres, T., Julià, R., Delgado, A., Llamas, F.J., Soler, V., Delgado, J., 2004a, Numerical dating algorithms of amino acid racemization ratios from continental ostracodes. Application to the Guadix-Baza Basin (Southern Spain), Quaternary Science Reviews , 23 , 717-730.
Ortiz J.E., Torres, T., Delgado, A., Julià, R., Lucini, M., Llamas, F.J., Reyes, E., Soler, V., Valle, M. (2004b). The palaeoenvironmental and palaeohydrological evolution of Padul Peat Bog (Granada, Spain) over one million years, from elemental, isotopic, and molecular organic geochemical proxies. Organic Geochemistry 35 (11-12), 1243-1260.
Ortiz, J.E. , Torres, T., Delgado, A., Reyes, E., Díaz-Bautista, A. (2009). A review of the Tagus river tufa deposits (Central Spain): age and palaeoenvironmental record. Quaternary Science Reviews 28, 947-963.
Sohn, I.G. (1958). Chemical constituents of ostracodes: some applications to paleontology and paleoecology. Journal of Paleontology 32, 730-736.
Torres, T. Cobo, R. Canoira, L. García Cortés, A. Grün, R. Hoyos, M. Juliá, R. Llamas, J. Mansilla, H. Quintero, I. Soler V., Valle, M. Coello, F.J. García- Alonso, P. Guerrero, P. Nestares, T. Rodríguez-Alto, Barettino, D. (1995). Reconstrucción paleoclimática desde el Pleistoceno medio a partir de análisis geocronológicos e isotópicos de travertinos españoles. Area B: Travertinos fluviales de Priego (Cuenca). Technical report, ENRESA, Madrid, Spain.
Torres, T., Llamas, J.F., Canoira, L., García Alonso, P., García Cortés, A., Mansilla, H. (1997). Amino Chronology of the lower Pleistocene deposits of Venta Micena (Orce, Granada, Andalousie, Spain). Organic Geochemistry, 26, 85-97.
Torres, T., García-Alonso, P., Canoira, L., Llamas, J.F., 2000, Aspartic Acid Racemization and Protein Preservation in the Dentine of European Bear Teeth, in Perspectives in Amino Acids and Protein Geochemistry (eds. G.A. Goodfriend, M.J. Collins, M.L. Fogel, S.A. Macko, J.F. Wehmiller), 349-355, Oxford University Press New York.
Torres, T., Ortiz, J.E., Llamas, J.F., Canoira, L., Juliá, R. y García de la Morena, M.A., 2002, Bear Dentine Aspartic Acid Racemization Analysis, Proxy for Pleistocene Cave Infills Dating, Archaeometry, 44(3), 417-426.
Torres, T, Ortiz, J.E, García de la Morena M.A., Llamas F.J, Goodfriend G., 2005. Aminostratigraphy and aminochronology of a tufa system in Central Spain. Quaternary Internacional 135, 21-33.
Wehmiller, J.F., Emerson, W.K., 1980, Calibration of amino acid racemization in late Pleistocene mollusks: results from Magdalena Bay, Baja California Sur, Mexico, with dating applications and paleoclimatic implications, The Nautilus, 94(1), 31-36
