Application of Multimolecular Biomarker ... - ACS Publications

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Chapter 13

Application of Multimolecular Biomarker Techniques to the Identification of Fecal Material in Archaeological Soils and Sediments Richard P. Evershed and Philip H. Bethell School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, United Kingdom This paper focuses on the chemical analysis of organic residues in archaeological soils, particularly on the development and application of lipid "biomarker" techniques for the detection and characterization of disaggregated fecal matter. The work has been directed towards the study of soils from experimental and archaeological sites to assess the possibility of employing "biomarkers" characteristic of feces. Initial efforts in this area focused on building on the principle that 5β-stanols (which arise in feces from∆ -stenolsby microbial reduction in the gut) may be of use in assessing fecal inputs into archaeological soils and sediments. The analytical methods employed are based on the use of gas chromatography/mass spectrometry with selected ion monitoring (GC/MS-SIM) to provide a very sensitive and selective means of analyzing for the characteristic steroidal marker compounds. Enhanced selectivity can be achieved by use of GC/MS/MS employing selected reaction monitoring (SRM). The use of 5β-cholestan-3β-οl (coprostanol and its congeners) to identify the sites of ancient cesspits has been demonstrated. In an investigation of a set of soil samples taken at intervals across an experimental field only the variation in the concentration of 5β-stanols reflected manure addition, together with the dynamic effects of soil erosion and possibly bioterbation. This paper summarizes our recent findings in this area, placing special emphasis on developments in the use of multi-component mixtures of biomarkers, i.e., 5β-stanols and bile acids, to identify the origin of fecal inputs into soils with a high degree of specificity. 5

Aged feces , either in the form of intact coprolites or dispersed in soil, have attracted considerable attention as a result of their potential to yield unique archaeological information relating to early human behavior, diet, parasites, disease, ecological adaptation, resource utilization, etc. (1-3). Intact fecal remains have been found in arid caves in desert areas, in intact cadavers, such as mummies and bog bodies, and in other extreme preservation environments such as frozen or waterlogged deposits. In temperate regions fecal remains are more commonly encountered in a disaggregated

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© 1996 American Chemical Society

In Archaeological Chemistry; Orna, M.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.



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Identification of Fecal Material

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state, concentrated in such features as latrines, cesspits and midden deposits, or widely scattered, as in the case of ditch-fills or manuring. Established methods of studying fecal remains include mainly the use of microscopy to identify morphologically intact fragments of food, e.g., plant fibers, pollen, bone fragments, and fish scales as well as parasite eggs and body parts, and inorganic materials, e.g., grits, derived from the tools or vessels used in the preparation of food (2, 4, 5). Chemical analysis of aged fecal remains, until recently, received only scant attention. Analyses for inorganic ions were among the first chemical analyses to be performed with a view to gaining dietary information (6). Problems exist in drawing conclusions from analyses of fecal remains recovered from waterlogged sites due to the dissolution of many inorganic components. In more recent years, the preservation of the organic constituents of ancient fecal remains has been demonstrated by use of GC/MS. Probably the most detailed study reported to date is that of Lin et al. (7) who investigated the survival of steroidal components in 2000-year-old human coprolites from dry deposits in Lovelock Cave, Nevada. All six samples that were analyzed using gas chromatography (GC) contained steroidal compounds. Although the concentrations of sterol, stanol and bile acids were somewhat lower than those of fresh human stool, the relative abundances of the individual components were very similar. In another study of cave coprolites from Danger Cave and Glen Canyon, Utah, amino acids, bile acids and lipids were readily detected (8, 9). The GC analyses performed in this latter study revealed no significant differences in the distributions of various lipids compared to desiccated modern samples. The potential for the chemical analysis of coprolites has also been noted by Wales and Evans (10). While these findings are important in demonstrating the survival of fecal lipids in intact coprolites, substantial scope exists for the investigation of lipids from disaggregated feces in soils. Earlier examples include the study of the contents of the fill of a ditch associated with a Roman fort at Bearsden, Scotland. Conventional palaeobotanical and zoological techniques were used in conjunction with GC and GC/MS (77). Different strata of the ditch fill were distinguished by their sterol content. One layer was deemed to represent a sewage deposit based on the detection of coprostanol, 5p-cholestan-3p-ol (1; for structures see Figure 1) and its C28 (2) and C29 (3) homologs and related oxo-steroids, and bile acids (e.g., 4 and 5). The finding of coprostanol was taken to be an indicator of human fecal material, and hence, the use of the ditch as a latrine drain. Pepe and co-workers (72, 13) analyzed archaeological sediments for their lipid content. Their findings of coprostanol in high abundance in one horizon was taken to indicate cess deposition. Related studies of lipids in soils and sediments include the analysis of fatty acids from an Eskimo midden, which provided evidence consistent with a high input of debris from marine animals (14), and the analysis of fatty acids in soils from a buffalo jump site in Canada (75). The above studies have established that specific chemical components, notably the steroidal compounds that occur ubiquitously in feces, possess the necessary chemical stability to survive for several millennia in a range of contrasting environments that are encountered at archaeological sites. Even where the physical evidence of feces is heavily degraded, e.g., through acid soil conditions, or is widely distributed as in the case of manuring, the detection of these specific markers should allow the location of fecal deposition. Our research has concentrated on the development of the application of biomarkers to the detection of disaggregated fecal matter in archaeological soils, with the following specific aims: (1) to assess the usefulness of different classes of chemical substance as indicators of inputs of fecal matter into archaeological soils and sediments; (2) to provide effective analytical methods for the detection of specific biomarkers in soils and sediments; (3) to identify the sites of ancient latrines, cesspits and midden dumps; (4) to distinguish human fecal matter from that of other animals; (5) to



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Archaeological Soil or Sediment Hexane Dissolution Saponification and Acid/Neutral Separation Diazomethane

Bile Acid Methyl Esters

Total Lipid Extract

Crude 5p-Stanols

TLC

BSTFA

Stanols and Sterols

Trimethylsilylation

BSTFA HDMS/ TMCS

Trimethylsilylation

GC/MS/MS SRM

Trimethylsilylation GC/MS SIM GC/MS SIM Figure 2. Scheme of extraction of fecal steroid biomarkers from archaeological soils. Abbreviations: BSTFA = A/;0-bis(trimethylsilyl)trifluoroacetarnide; HMDS = hexamethyldisilazane; TMCS = trimethylchlorosilane. (See text for all other abbreviations.)



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distinguish fecal matter from decayed vegetable matter; and (6) to establish sites of animal penning enclosures or stables. Our aim is therefore to be able to demonstrate the survival of molecular markers of fecal matter in soils and sediments at archaeological sites. Although the primary goal of this investigation was to develop the use of lipid biomarkers, particularly steroidal compounds, as indicators of fecal matter inputs into archaeological soils and sediments, throughout these studies we have compared and contrasted the information provided by lipid biomarkers to that derived from other techniques that are used in the study of soils and sediments at archaeological sites. Where comparisons have been drawn they will be referred to in the appropriate sections below. (See also Bethell, P., et al. submitted for publication to J. Archaeol Science). Analytical Method Development The analytical protocols employed in this work focused primarily upon the use of 5βstanols and bile acids (e.g. 1-5) preserved in archaeological soils and sediments. The overall analytical scheme used to assesses archaeological soils and sediments for the presence of these compounds is summarized in Figure 2. More detailed accounts containing the precise practical information can be found in References 16-18. Although long-chain alcohols, fatty acids, alkanes and wax esters were the major components of the soils studied these are believed to represent the natural background input from plants, fungi and microorganisms associated with the soil. The 5β-8ΐ^ο1δ and bile acids were chosen as biomarkers for use in this work due to their ubiquitous occurrence in mammalian feces and the fact that their structures can be used diagnostically to determine their original source with a high degree of certainty. Moreover, these compounds are not the normal products of the decay of plant or animal matter in the soil. Although 5β-8ΐ3ηο1δ and bile acids occur only in very low concentrations in soils and sediments the use of combined GC/MS/SIM affords the high sensitivities and selectivities necessary to detect the target compounds in the majority of archaeological and experimental materials studied. The electron ionization (70 eV) mass spectra of the most commonly occurring animal and plant derived Δ5stenols and 5β-8ί^ο1δ are shown in Figure 3 and the ions used to detect characteristic marker compounds (stanols and stenols) are listed in Table I. Specific examples of the application of these techniques are discussed in the remainder of this paper. An alternative approach that has been explored uses the new technique of tandem mass spectrometry, specifically GC/MS/MS using selected reaction monitoring (SRM). Figure 4 shows the product ion spectrum produced by the collision induced dissociation of the m/z 370 ion ([M-TMSOH]" ") of coprostanol in the collision cell of a triple stage quadrupole mass spectrometer. The decomposition, m/z 370 -* 215, which arises via cleavage across the D-ring of the steroid nucleus, accompanied by a proton transfer, raises possibilities for the highly selective detection of 5β-8ΐ3ηο1δ by GC/MS/MS employing SRM as shown schematically in Figure 5. Indeed in practice this new technique was found to have considerable potential for the trace analysis of 5βstanols, allowing highly selective analyses with a significantly simplified sample preparation strategy to be adopted compared to that required when conventional GC/MS was employed. The hexane-soluble fraction of the total lipid extracts (chloroform/methanol, 2:1 v/v) were trimethylsilylated and analyzed directly by GC/MS/MS, using a triple stage quadrupole instrument, without further purification. Although the hexane soluble fraction comprises a complex mixture of cyclic and acyclic lipid species 5β-δίίΐηο1δ (and 5a-stanols) were readily detected with high selectivity. The use of the method was demonstrated in the analysis of 5 β - 8 ί 3 η ο ΐ 8 in various archaeological and experimental agricultural soils. The method greatly simplifies 1



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Time (m) Figure 6. Partial GC/MS SIM (m/z 215) chromatograms of an extract of a soil horizon from a Late Saxon/Medieval garderobe from West Cotton, Northants, U.K. IS is an internal standard, 5p-pregnanol, which is added at the extraction stage (see Figure 2) for quantification purposes.


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Molecular Markers of Manuring The identification of evidence of manuring practices from archaeological sites has great potential. Such evidence could enable identification of site limits, field systems, development of early agriculture systems, settlement/farmland relationships, etc. The analysis of animal feces and manure for reliable, characteristic molecular biomarkers in the soil, and the subsequent measurement of the abundances of those biomarkers in the soil, formed the basis of this phase of our investigations. This approach was chosen as certain stable molecules are known to be more resistant to decomposition over long periods of time, and are more likely to be retained in the soil than other chemical evidence of manuring. Initial alterations in the soil organic matter content are not thought to survive long beyond the end of the manuring episode, and other criteria, such as phosphorus enhancement, are very dependent on soil conditions, and have rarely been subject to rigorous analysis in the past. The analyses performed here were carried out on modern experimental material from the Butser Hill Farm site in Hampshire, U.K., where a plot under wheat cultivation had been subject to manuring over half its area for 13 consecutive years; the abundances of various biomarkers (specifically sterols and 5p-stanols) were determined in the manured and non-manured areas. The analyses were again based upon extraction of the lipids from the soil samples followed by fractionation by thin layer chromatography and analysis by GC and GC/MS. A distinct enhancement of specific biological molecules characteristic of cattle feces or manure, predominantly 5p-stigmastanol (3) was found in the manured area when compared to the non-manured area as shown in Figure 7. The method thus was found to have great potential for archaeological application. The results from the lipid work were also compared and contrasted with those from magnetic susceptibility measurements, total lipid and elemental (C, H and N) analyses, highlighting the advantages of the biomarker approach. A more detailed discussion of these results is given in reference 18. It is worth noting the near gaussian distribution observed for the 5p-stanols across the field which represents a departure from the theoretical distribution resulting from soil erosion and bioterbation effectively "blurring" the boundary between the manured and non-manured areas of the experimental plot. Detection of Bile Acids in Archaeological Soils and Sediments The bile acids found in higher animals are biosynthesized directly from cholesterol by saturation of the double bond, epimerization of the 3p-hydroxyl group, introduction of hydroxy Is into the 7a and 12a positions, and oxidation of the side chain to a C-24 carboxylic acid. Bile acids act as detergents in the emulsification of dietary fats. Although the primary bile acids in mammals are cholic and chenodeoxycholic, they are transformed in the intestine by microorganisms into the secondary bile acids, lithocholic (4) and deoxycholic (5) acids. Of significance archaeologically is the fact that the formation of bile acids is the most important pathway for the metabolism and excretion of cholesterol in mammals. The rate of excretion of bile acids in human feces is of the order of 0.5 g/day. The analytical protocol that was adopted for the analysis of bile acids is summarized in Figure 2. Analyses were performed on the soil samples recovered from a modern latrine and the archaeological soils and sediments already discussed above in relation to the study of 5p-stanols. The results we obtained concur with those of earlier studies (77) and confirm that bile acids survive in archaeological soils. Figure 8 shows the total ion chromatogram obtained by GC/MS-SIM for the bile acid fraction of the total lipid extract of soil from a Late Saxon/early medieval garderobe.



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Figure 7. Histogram showing the relative abundance distribution for 5p-stanols in soils samples taken at meter intervals across the cultivated field at Butser Hill Experimental Iron Age Farm compared to control soils taken away from the cultivated area.



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5P-Stanols Present No No Fecal Above Control Matter Detected Concentration Yes Human or Porcine Fecal Matter

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Hyodeoxycholic and Hyocholic Acids Present

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Human Fecal Matter

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Ruminant Fecal Matter

Yes Τ Porcine Fecal Matter Figure 9. Differentiation of fecal inputs to archaeological soils by a multimolecular approach based on the combined use of 5β-8ί^ηοΐ8 and bile acids.



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The relative proportions of the lithocholic and deoxycholic acid components is consistent with that seen in modern latrine soil. The hyocholic acid (6) component at longer retention time is an internal standard added for the purpose of quantification. The results obtained from the range of sample examined confirm the widespread survival of bile acids in archaeological soils and sediments and as such extends the possibilities for using steroid biomarkers as indicators of fecal inputs into archaeological soils. In common with our findings for 5|3-stanols in the various control soils examined there are indications that these compounds also survive widely in the environment and as such represent a new class of indicators of fecal matter (pollution) in the ancient and modern environment alikerMeasurement of the relative proportions of different sterols and bile acids in individual coprolites has been tentatively proposed as a means of determining the relative contribution of plant and animal foodstuffs to the diet (7). The composition of the fecal bile acids also vary with the health or disease state of individuals. However, substantial research is required before such principles can be applied with confidence. The complementary use of 5p-stanols and bile acids as multi-molecular markers in the study of disaggregated fecal matter is discussed further below. Multi-Molecular Markers for Determining the Sources of Fecal Inputs to Archaeological Soils It is clear from the results of the various analyses of contemporary reference and archaeological materials summarized above that possibilities exist for distinguishing between the sources of different fecal inputs into soils at archaeological sites on the basis of their steroid biomarker content. For example, a distinction can be drawn between fecal inputs from various animals, e.g., humans (omnivores) vs. ruminants (herbivores), due to the differences in the relative proportions of coprostanol (1) and 5p-stigmastan-3p-ol (3) present. In the case of humans the relative proportion of coprostanol:5p-stigmastan-3p-ol is approximately 5.5:1, while in the case of ruminants (sheep and cows) the proportions are reversed, i.e., the ratio of coprostanol:5βstigmastan3p-ol is about 1:4. However, drawing a distinction between the feces originating from two omnivores is less straightforward since the fecal 5p-stanols of both humans and pigs are dominated by coprostanol. However, a clear distinction can be drawn between human and porcine fecal matter by consideration of the bile acids they produce. As discussed above, lithocholic (4) and deoxycholic (5) acids are the dominant bile acids in the feces of healthy humans. Although lithocholic acid is present in porcine feces, the high abundance of two bile acids that are insignificant in healthy humans, namely, hyodeoxycholic (6) and hyocholic (structure shown in Figure 8) acids, enables a clear distinction to be drawn between the two. We have termed this method of distinguishing between the origins of fecal matter through the combined use of two different classes of steroid biomarkers as a multi-molecular approach and have summarized it schematically in Figure 9. Acknowledgments In presenting this paper we should like to acknowledge the contributions of all our collaborators and the experts that have contributed to the success of this research, In particular we should like to note the contributions of the late Dr. Jim Ottaway for his enthusiasm and encouragement in the early stages of this work and for provision of samples. Dr. Peter Reynolds is thanked for provision of samples and Drs. Gillian Campbell and Mark Robinson of Oxford Museum are thanked for their expert advice and assistance with sampling from the Raunds Area Project; Dr. Nick Walsh who is also thanked for assistance with inductively coupled plasma atomic emission


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spectroscopy analyses of the soils from the Butser Experimental Iron Age Farm and Mr. Mark C. Prescott for assistance with the GC/MS analyses.


Literature Cited 1. Shackley, M. Using Environmental Archaeology; Batsford: London, 1985. 2. Fry, G. F. In The Analysis of Prehistoric Diets; Gilbert, R. I.; Mielke, J. H., Eds.; Academic Press: London, 1985; pp 127-154. 3. Hillman, G. C., In Lindow Man: The Body in the Bog; Stead, I. M.; Bourke, J. B.; Brothwell, D., Eds.; British Museum: London, 1986; pp 99-115. 4. Heizer, R. F.; Napton, L. K. Science 1969, 165, 563-568. 5. Bryant, V. M.; Williams-Dean, G. Scientific American 1975, 232 (1), 100-109. 6. Wakefield, E. G.; Dellinger, S. C. Annals of Internal Medicine 1936, 9, 14121418. 7. Lin, D. S.; Conner, W. E.; Napton, L. K.; Heizer, R. F. J. Lipid Res. 1978, 19, 215-221. 8. Napton, L. K.; Heizer, R. F. In Archaeology and the Prehistoric Great Basin Subsistence Regime as Seen from the Lovelock Cave, Nevada; Heizer, R. F.; Napton, L. K., Eds.; Contributions of the University of California Archaeological Research Facility No. 10; University of California at Berkeley Press: Berkeley, 1970. 9. Fry, G. F. In Miscellaneous Paper No. 23, University of Utah Anthropological Papers No. 99 (Miscellaneous Collected Papers 19-24); University of Utah Press: Salt Lake City, 1978. 10. Wales, S.; Evans, J. In Science and Archaeology - Glasgow, 1987: Proceedings of a Conference on the Application of Scientific Methods to Archaeology; Slater, Ε. Α.; Tate, J. Α., Eds.; Oxbow: Oxford, 1987; pp 403-412. 11. Knights, Β. Α.; Dickson, C. Α.; Dickson, J. H; Breeze, D. J. J. Archaeol. Science 1983, 10, 139-152. 12. Pepe, C.; Dizabo, P. Revue d'Archaeometrie 1990, 14, 23-28. 13. Pepe, C.; Dizabo, P.; Scribe, P.; Dagaux, J.; Filiaux, J.; Saliot, A. Revue d'Archaeometrie 1989, 13, 1-11. 14. Morgan, E. D.; Cornford, C.; Pollack, D. R. J.; Isaacson, P. Science and Archaeology 1973, 10, 9-10. 15. Dormar, J. F.; Beaudoin, A. B. Geoarchaeology 1989, 6, 85-98. 16. Bethell, P. H.; Evershed, R. P.; Goad, L. J. In Prehistoric Human Bone: Archaeology at the Molecular Level; Grupe, G.; Lambert, J. B., Eds.; Springer Verlag: Berlin, 1993; pp 229-255. 17. Bethell, P. H.; Goad, L. J.; Evershed, R. P.; Ottaway, J. J. Archaeol. Science 1994, 21, 619-632. 18. Bethell, P. H.; Evershed, R. P.; Reynolds, P. J.; Walsh, P. J. J. Archaeol. Science 1996, 23, in press. 19. Rosenfeld, R. S.; Fukushima, D. K.; Hellman, L.; Gallagher, T. F. J. Biol. Chem. 1954, 211, 301-311. 20. Readman, J. W.; Preston, M . R.; Mantoura, R. F. C. Marine Pollution Bulletin (Reports) 1986, 17, 298-308. RECEIVED

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