Variability in the preservation of the isotopic composition of collagen from fossil bone

Geochimica et Cosmochimtca ACIa Vol. 52, pp 929-935 Copyright © 1988 Pergamon Press pic Pnnted In USA

0016-7037/88/$3.00

+ .00

Variability in the preservation of the isotopic composition of collagen from fossil bone NOREEN TUROSS, MARILYN L. FOGEL and P. E. HARE Geophysical Laboratory, Carnegie Institution of Washington, Washington, D,C. 20008, U,SA

(Received July 30, 1987; accepted in revised/arm January 27, 1988) Abstract-Collagen from bone was prepared by several methods. For modem and well-preserved bone the Ol3C and Ol5N ofcollagen replicas obtainedafterHO or EDTA demineralization weresimilarto thoseobtainedwitha gelatinization procedure.However, in more poorlypreserved fossil bone the Ol3C and 015N variedamong the differentprotein extracts. The yield of collagen obtained with EDTA demineralization was consistently higher than extraction procedures that used HCI. The Ol3C of individual amino acidsseparatedfrom the collagen of modem and fossil whalebone varied up to 17%0, and the Ol5N from the same amino acids ranged over 47%0. The Ol3C and Ol5N of most amino acids clustered closely to the average of the HCI insolublecollagen. The Ol3C of the major amino acid in collagen, glycine, differed from the average HCI insolublecollagen by approximately 8%0 in the fossil whale and 14%0 in the modem whale. The Ol5N of glycine differed from the average HCI insolublevaluesby approximately 40/00 in the fossil whaleand 70/00 in the modem whale, Thus, diagenetic changesthat alter the ratio of glycine to other amino acidsin bone can be expectedto perturb the valuesfor carbon and nitrogen isotopes. INTRODUCTION

generated in the isolation of protein from bone (DENIRO, 1985). Carbon/nitrogen values of 2.9-3.6 from gelatinous extracts of bone are thought to be indicative of collagen with diagenetically unaltered carbon and nitrogen values. The assumptions involved in equating elemental analysis with isotopic preservation are: I) gross impurities that might contribute carbon or nitrogen to the extract will shift the elemental analysis outside of the acceptable range, 2) the fractionation of the carbon and nitrogen isotopes are uniform throughout the amino acids of the collagen molecule, and 3) all of the carbon and nitrogen measured derives from the collagen molecule. The noncollagenous proteins in bone, which may number as many as 200 (DELMAS et al.. 1984), are generally ignored in stable isotope studies. Because virtually all noncollagenous proteins in bone are soluble after demineralization and present in small « 10% oftotal protein) amounts, these proteins do not contribute significantly to the overall isotopic pattern derived from bone, either in traditional gelatin or replica preparations. Animals do not fractionate each amino acid uniformly into the collagen molecule ofbone. The ol3 and 015N values of collagen are a composite of the fractionation of isotopes into each of the individual amino acids (HARE and ESTEP, 1983). When the 015N and Ol3C of individual amino acids from denatured bovine Achilles tendon and bison fossil collagen were compared, the essential amino acids were found to deviate from the HCl insoluble collagen average. In addition, glycine was found to be enriched in l3C and depleted in 15N relative to the total insoluble protein isotopic values. In this study the isotopic variability among the individual amino acids ofcollagen from the bone ofa modern and fossil whale is compared. Because the amino acid glycine makes up a large percentage of the total amino acid composition of collagen (approximately 30%), changes in the glycine content of fossil protein will be reflected in the diagenetically altered collagen Ol3C and Ol5N values. Several extraction techniques are compared in order to assess the purity, yield and isotopic composition of the insoluble material. The general collagen extraction procedure used in Ol3C and Ol5N analysis is based on techniques devel-

A BASIC ASSUMPTION in the stable or radiometric isotope analysis of fossil bone is that the biological isotopic signal is maintained through diagenesis and the subsequent extraction for chemical analysis. Collagen, the major protein in bone, is thought to retain l3C;12C and 15N;t4N values postmortem (VAN DER MERWE, 1977; SCHOENINGER et al., 1983; SCHWARCZ et al., 1985) even though fossil collagen is known to degrade with time after death (WYCKOFF, 1973; TUROSS, 1980; HARE, 1980). Carbon isotope values (ol3e) have been used to assess the contribution of maize to the prehistoric diet (VAN DER MERWE, 1982) and to distinguish marine from terrestrial feeders (CHISHOLM et al.. 1982). More recently investigators have combined carbon and nitrogen isotope data in order to interpret the marine versus terrestrial food sources in prehistoric diets (SCHOENINGER et al., 1983; DENIRO, 1987). Two fundamental questions pervade the use of stable isotope data in paleodiet reconstruction: (I) Are complex, heterogeneous diets reflected in carbon and nitrogen isotopes of bone collagen such that paleodiet interpretation is possible? and (2) is the biological signal in the carbon and nitrogen isotopes of bone organic material altered in the decay, burial, and fossilization process? The use of 015N to predict the contribution of marine sources to a diet has been questioned (HEATON et al., 1986). The effect of annual rainfall on plant source nitrogen isotopic fractionation has been raised. It has also been suggested that nitrogen utilization in water-stressed animals needs to be considered (AMBROSE and DENIRO, 1987). Because the answer to these and other questions involving complex diets and ecological niches will require the comparison of many stable isotope values from different laboratories, it is imperative that an isotopically unaltered biological source material be analyzed. Protein diagenesis in fossil bone must be examined closely to ascertain when stable isotope values differ from the original biologically controlled isotopic fractionations. The integrity of the collagen molecule in fossil bone has been studied by measuring the amount of carbon and nitrogen

e

929

N. Tuross, M. L. Fogel and P. E. Hare

930

oped for radiocarbon dating (LONGIN, 1970). The usefulness of a gelatinizing procedure is compared to demineralizing protocols that rely on the presence of an insoluble collagen replica after the mineral has been removed. The gelatin extracts from some fossil bones have been shown to contain a range and distribution of amino acids found in collagen from modem bone (NELSON et al., 1986). However, some fossil bones are known to have amino acid patterns that have been altered diagenetically (HARE, 1980). We examine how such changes in amino acid profile, particularly glycine depletion, could alter stable isotope values. MATERIALS AND METHODS

Sample source Modern bovine fetal calvaria were obtained at slaughter. Modern sperm whale rib was collected at autopsy after the whale had beached less than twenty-four hours previously. These samples were transported on dry ice and stored at -80°C until use. Fossil whale bones were obtained from G. Miller (lNSTAAR). Both whales were excavated in a recently eroded section of Forlandsundet, on the west coast of Spitzbergen, Norway. INSTAAR #3 DAU 7, was radiocarbon dated (GX-10593) at 9735 ± 160 years B.P. Another whale, INSTAAR #M79-SB2, contained less radiocarbon activity than background at two laboratories (GX-799-G and QL-1693). The skeleton was excavated approximately 90 km north of 3 DAU 7. The estimated age of this fossil bone based on evidence in the surrounding sedimentary unit is 75,000 ± 15,000 years B.P. (GIFFORD MILLER, pers. commun.). Both whale bones were excavated from permafrost and the fragmentary pieces could not be identified as to genus. Several human bone fragments were from the San Diego Museum of Man collection. These samples were excavated in the 1920s by Malcolm Rogers and were obtained from Rose Tyson of the San Diego Museum of Man. These fossil bones were generally found in sandy soil in a variety of southern California locations. The Moa bird bones were collected by N. Tuross in a limestone cave in New Zealand. No radiocarbon date has been obtained for these bones, however this sample must exceed 300 years B.P. as this large flightless bird has been extinct since that time. Two additional bones of a zebra and wildebeest were obtained from K. Behrensmeyer of the Smithsonian Institution. They are of unknown age and were surface collected in Kenya. These fossil bones from Kenya show extensive mineral replacement of the original apatite. All ofthe bones described above, with the exception of the modern bovine and whale specimens, are considered fossil bones. The fossil bones were designated well, moderately or poorly preserved based on the total bone % protein relative to modern controls: >50%, well preserved; > 10%, moderately preserved; and < I0% poorly preserved.

Protein isolation Bone samples (50-300 mg) were extracted as described in SCHOENINGER and DENIRO(1984) and DENIRO (1985). Briefly,bone was powdered; demineralized for 20 minutes in 1.0 M HCI at room ~emperature; filtered through a sintered glass filter; washed and stirred In 0.125 M NaOH for 20 hours; washed to constant pH and solubilized in I mM HCI for 10 hours at 90°C. The final product was again filtered through a sintered glass filter and lyophilized. This extract will be referred to as gelatin. Bone chunks (50-300 mg) were demineralized in 50 ml of 1.0 M HCI at 4°C with gentle shaking for 24 hours. When a collagen replica was obtained, the residue was washed 10 times with 50 mls of glass distilled water and lyophilized. This product will be referred to as HCI insoluble collagen. A similar demineralization procedure was performed on bone pieces by stirring 50-300 mg chunks of bone in 50 ml of 0.5 M EDTA, pH 7.2, at 4°C for varying amounts of time up to five days. After all mineral had been removed, as evidenced by a translucent. pale yellow collagen replica, the residue was washed 15 times with

glass distilled water and lyophilized. This product will be referred to as EDT A insoluble. The possibility of contaminating EDTA was monitored by ammonia levels in the amino acid analysis as described below. All EDTA insoluble residues had less than I ng EDTA contamination per mg of dry protein. The yield of protein obtained by these extraction procedures was calculated by dividing the dry weight of the residue obtained by the dry weight of the starting bone sample. This figure was then corrected for total possible yield from a given fossil bone based on the micromoles of amino acids per mg of bone. For example, if a fossil bone contained 15% protein by whole bone amino acid analysis (modern bone contains approximately 30% protein), and 7.5 mg dry weight residue was obtained from a 100 mg bone fragment, this was designated as a 50% yield of total recoverable protein. Each sample was extracted twice by the above protocols and the reported yields are an average of the two extractions. The micromoles of amino acids per milligram dry weight of bone are also given. This value has not been corrected for loss during hydrolysis. Yields of replicate analyses of demineralization with HCl or EDTA varied less than 10%. Replicate gelatinization extracts varied from 12% to 300% with the variability in extract weight inversely proportional to amino acid content of the whole bone.

Amino acidanalysis Whole bone and each type of extracted residue were analyzed for amino acid composition. Approximately 10 mg bone and I mg residue were hydrolyzed in 6 N HCI under N 2 at 110°C for 20 hours. The amino acid compositions were determined as described by HARE (1977). Detection of the ninhydrin complex was monitored at 440 nm and 570 nm. Glycine/aspartic acid values of 5.5-6 are representative of collagen from modern bone (HARE, 1980).

Separation of individual aminoacids for isotope analysis Insoluble collagen was prepared from HCI demineralized bone. Milligram quantities of individual amino acids were separated as previously described (MACKO et al.. 1987; HAREand ESTEP, 1983). This preparative cation exchange system uses only HCl as an eluent (0.8-4.0 N HCI). Purity of the amino acids was determined by a complete amino acid analysis as described above.

Isotopic analysis Stable isotope analyses were performed as previously described (ESTEP and VIGG, 1985). Briefly, 1-5 mg of individual amino acids. 5 mg of bone extract or 30-50 mg of whole bone were combusted in a sealed quartz tube at 900°C for I hr. The combustion tube was cooled at a defined rate, and the products of combustion were isolated by cryogenic distillation. Isotope ratios are reported in terms of 0:

ol3C or I IN =

Rsample

-I

X

103

Rstandard

where R = l3C/ 12C or IIN/ 14N. Standard deviation for replicate analysis of the bone extracts is ±O.I %0 for Ol3C and ±0.2%0 for OlIN. For individual amino acids, the average difference between the isotopic composition of the standard amino acids before and after chromatography was ±0.3%0 for 013C and ±0.4%0 for OlIN.

RESULTS

Individual amino acid rPc and rPN from bonecollagen The isotope ratios of carbon and nitrogen in individual amino acids from collagen of fossil and modem whale bone are variable. The pattern of deviation of individual amino acids from the collagen average are similar in the modem and fossil bone collagens. The o13e and 015N of glycine, serine and threonine differ significantly from the remaining amino acids tested (Figs. 1 and 2). In general, glycine, serine and

931

Isotopic composition of fossil bone collagen Table

1.

l l Stable isotope values (6 3C and 6 5N) of modern and foss 11 whale bone amino acids

o

Hodern whale

Fossil whale*

-5 HCl insoluble

~- -10

Average measured amino seidsn

Glycine

o

... M

I COLLAGEN

~---~-~~~~~~

-15

00

-20

WHALE

-25 '----l_----'-_--'-_L-----'-_-'--_-'------'_----'--l VAL PRO HYF GLU ASP ALA GLY SER TIIR AMINO ACIDS

FIG. I. Individual amino acid Ol3C from hydrolysates of HCl insoluble collagen of fossil whale bone (INST AAR#M79-SB2) and modem sperm whale bone. Dashed lines indicate the Ol3C ofthe total HCI insoluble residue.

threonine values for Ol3C in the modem and fossil whale were more positive than the average value of an EDTA insoluble residue whose amino acid composition was virtually identical to purified collagen. The glycine, serine and threonine values of 015N were significantly more negative than that ofthe total extract. The Ol3C and 015N of glycine relative to all other measured individual amino acids and the HCl insoluble collagen is shown in Table I. While only nine amino acids (eight in the case ofthe modem whale) were isolated in sufficient quantity for isotopic analysis, these nine amino acids contribute 79.2% of the carbon and 77.9% of the nitrogen to the collagen molecule. A weighted average of the Ol3C data for the nine amino acids from the fossil whale was used to calculate the remaining 20% of the carbon which would have an average of Ol3C of - 21.4. This value is slightly more negative than the average of all of the measured amino acids excluding glycine, serine and threonine (O l3C = -19.2%0). Thus, the collagen stable isotope value for carbon is made less negative by the glycine, serine and threonine values. In the case of nitrogen isotopic 25 ,--,c------r----.--....,--,.--,------,----,--.,.-----, MODERN WHALE

15

10 L COLLAGENFOSSIL

5 "~ 0 - -5 :z;

~

eo

-10 -15 -20

~::r,

VAL PRO HYP GLU ASP

*IN8TAAR #M79-8B2 Estimated age is 75,OOOf15,OOO yrs. B.P. IIWeighted average based on the 1JDI01es of amino scids/mg dry weight of modern bone (Hare, 1980).

fractionation, the contribution of glycine, serine and threonine to the total collagen 015N is even more pronounced. The amino acids that were not separated would have an average calculated 015N value of ± 14.8%0. This number is within the range of the clustered 015N values of the amino acids valine, proline, hydroxyproline, glutamic acid, aspartic acid, and alanine. A loss of glycine relative to other amino acids in collagen would have the effect of making the average 015N value more positive and the average Ol3C value more negative. Loss of glycine would not be equally reflected in the two isotopic ratios. Because glycine contributes approximately 30% ofthe nitrogen to collagen, but only approximately 18%of the carbon, any loss of glycine would have nearly twice the effect of the observed 015N as compared to ol3e. Loss of serine and threonine would change observed collagen 015N and Ol3C values in the same direction as loss of glycine. However, serine and threonine make up a very small percentage ofthe overall nitrogen and carbon in collagen. Thus, even total loss ofserine or threonine amino acids would have a small effect on the carbon and nitrogen isotope ratios.

Collagen extraction from modern andfossil bone Collagen can be extracted from modem samples either as a replica after HCl or EDTA demineralization or by the more Table 2. Protein chemistry, yield and isotopic composition of modern bones Sample (% Protein : amino acid concentration)* Extraction Method

6l5 N

6l3 C Yield(%)

+5.9 +6.4 +6.0 +6.0

-17.6 -17.3 -18.3 -18.4

Sperm Whale (74% : 1. 6IJ111oles/mg) +10.2 Whole Bonell HCl Insoluble +10.9 EDTA Insoluble +10.4 +9.8 Gelatin

-14.4 -18.7 -19.0 -13.7

Fetal Bovine (100% : 2.1IJ111oles/mg) Whole Bone HCL Insoluble EDTA Insoluble Gelatin

20

ALA GLY SER

1J TIIR

AMINO ACIDS

FIG. 2. Individual amino acid 015N from hydrolysates of HCl insoluble collagen of fossil whale bone (INSTAAR#M79-SB2) and modem sperm whale bone. Dashed lines indicate the 015N of the total HCI insoluble residues.

-14.4 +10.3 -17.3 +9.8 -12.6 +7.0 -14.7 +8.8 -0.8 +3.4 -9.4 +5.7

83 85 75

79 88 71

*The % protein for all samples was defined as micromoles of amino acids per dry weight of bone relative to the fetal bovine calvaria. The amino acid concentration is expressed as lJIIIoles of amino acid per mg dry weight of bone. The protein level found in modern sperm whale rib is less than the bovine bone due to the high level of lipids found in whale bone. IISperm whale bone extracted in chlorofYsm/methanol3(3/l) EDTA: 6 N=+0.8; 6 C=-34.5,

932

N. Tuross, M. L. Fogel and P. E. Hare

common gelatinization procedure (SCHOENINGER and DENIRO, 1984; DENIRO, 1985). The extracted collagen has isotopic values for nitrogen and carbon that are similar in these modern samples (Table 2). The amino acid patterns of these extracts all resemble collagen in possessing 90-110 residues/ 1000 of hydroxyproline and glycine/aspartic values that ranged from 5.9-7.0. Interference by lipids affected the ol3e values obtained from the modern sperm whale bone. Because the whole bone was extracted in chloroform/methanol (3:1) to remove lipids, and the gelatinized sample was mechanically disrupted, it is assumed that these ol3e values are closer to the true collagen value. In the category of well preserved fossil bones (Table 3), two whale bones that had been removed from permafrost contained good levels of collagen preservation. The whole bone and all three extracts show consistent values for both Ol5N and ol3 e. The difficulty observed with lipids in the modern whale bone was not seen in either fossil whale bone. An increased yield of collagen was obtained from the EDT A demineralized extracts. Two fossil bones were defined as moderately well preserved (Table 4). The amino acid composition of these two fossils resembled collagen, but the glycine/aspartic acid values for both bones had fallen to the 4-5 range. The glycine/aspartic acid values observed in the whole bone hydrolysates varied with repeated amino acid analysis. This variation may be due to differential preservation of the collagen molecules in the bone. All of the Ol5N and ol3e values observed for SDM 16709 were tightly clustered. Both Ol5N and ol3e values for this bone fall within the range of previously reported values suggested to be indicative of a diet heavy in marine sources (DENIRO, 1987). The difference in the Moa bird bone carbon isotopic composition of whole bone as compared to the demineralized extracts is most likely due to carbonate contamination ofthe inorganic phase. The ol3e for all the Moa bird extracts are quite consistent and in agreement with previously reported herbivore ol3e ranges (SCHOENINGER and DENIRO, 1984). One of the striking features of the group of bones labeled

Table 3. Protein chemistry, yields and isotopic composition of well preserved fossil bones Sample (% Protein : amino acid concentration)* Extraction Method Whale(83% : 1.7~oles/mg) Whole Bone HCl Insoluble EDTA Insoluble Gelatin

Held(%)

+11.6 +11. 2 +11.5 +11.5

-16.4 -16.8 -16.5 -16.4

+10.0 +9.7 +9.8 +10.4

-16.5 -17.1 -17.3 -17.6

70 82 57

Whalefl (51%-1.1~oles/mg)

Whole Bone HCI Insoluble EDTA Insoluble Gelatin

30 49 33

*See Table 1. for description. -Radiocarbon date (GX-l0593) 9735+160 yrs.B.P. #INSTAAR #M79-SB2: estimated age, 75,000 yrs. B.P. (see Materials and Methods).

Table 4. Protein chemistry, yields and isotopic composition of moderately preserved fossil bones Sample (% Protein

amino acid concentration)*

Extraction Method Human (SDM# 16709) (20% : 0.42~oles/mg) Whole bone HCl Insoluble EDTA Insoluble Gelatin

+19.5 +18.7 +19.6 +19.6

-14.6 -14.6

Moa (12% : 0.25~oles/mg) Whole Bone HCL Insoluble EDTA Insoluble Gelatin

+0.9 -0.5 -1. 0 -0.1

-14.8 -22.4 -22.4

-13.2

-13.7

40 66 12

37 54 10

-22.7

*See Table 1. for description. #SDM indicates the San Diego Museum of Man collection number.

poorly preserved (Table 5) is the yield in the gelatinization procedure of values equal to or greater than 100%. This phenomenon results not from actual protein yield, but rather from contaminating non-protein material brought through the extraction procedure. This can be seen in the amino acid analysis of the gelatinous residues where the percent dry Table 5. Protein chemistry, yields and isotopic compositions of poorly preserved fossil bones Sample (% Protein

amino acid concentration)*

Extraction Method Human (SDM 19254) (6.5% : 0.14~01es/mg) Whole Bone HCl Insoluble EDTA Insoluble Gelatin

+15.2 +16.0 +16.3 +15.4

-17.9 -17.0 -16.5 -18.0

22 55 137

Human (SDM 19244) (2.5% : 0.05~01es/mg) Whole Bone HCl Insoluble EDTA Insoluble Gelatin

+15.9 +10.0 +16.8 +18.4

-16.4 -21. 8 -18.3 -20.5

30 62 1000

Human# (SDM 16755) (0.09% : 0.002~oles/mg) Whole bone +15.5 Gelatin +10.8

-14.6 -24.7

160

Wildebeest fl (0.12% : 0.002~01es/mg) +7.5 Whole bone Gelatin -3.0

-0.6 -19.8

120

Zebra# (0.45% : O.Ol~oles/mg) Whole bone +11.6 Gelatin -2.3

+0.3 -25.5

100

*See Table 1. for description. Poorly preserved fossil bones were defined as samples with less than 10% of modern protein levels in which the following were observed: 1) diminished hydroxyproline levels relative to collagen and, 2) decreased glycine/aspartic acid ratios indicative of collagen alterations. #Total noncollagenous amino acid pattern as indicated for three of the samples above in which no hydroxyproline ia present in the bone, and glycine is no longer the predominant amino acid.

Isotopic composition of fossil bone collagen weight of the gelatin fraction that is made up of amino acids falls dramatically with decreasing total bone protein content (Table 6). Contaminants could contain either noncollagenous carbon or nitrogen. In addition, it is clear that production of a substantial residue from the traditional gelatinization procedure is not a criterion for integrity of the collagen in bone. The extracts of the two human bone samples (SDM 19254 and 19244) (Table 5) are quite different as to the relative consistency of their stable isotope values. In sample SDM 19254 (6.5% of modern protein levels) the glycine/aspartic acid value had declined to 5.5 in the whole bone, and this value remained essentially unaltered in the EDTA (5.6) and gelatinization (5.3) extraction procedures (Table 7). In contrast, for SDM 19244 (2.5% of modern protein levels) the glycine/aspartic acid values fell from 4.6 for bone and EDTA extract to 2.9 for the gelatinization procedure (Table 7). This fall in glycine/aspartic acid is reflected in the change of b1SN ofSDM 19244 EDTA extract from +16.8 to +18.4 in the gelatin. The increase in the b1SN in the gelatinization extract of this human bone could well lead to misinterpretation of dietary input. The major difference seen in both the carbon and nitrogen isotope ratios of the HCI insoluble extract of SDM 19244 may be due to humic acid contamination. Extraction with EDTA will remove humic and fulvic acid material under certain conditions, and this technique has been used successfully in treating peat derived bone and textiles (TUROSS, unpublished data). The SDM 16755, wildebeest and zebra samples listed in Table 5, have noncollagenous amino acid patterns ti.e., no hydoxyproline and reduced glycine/aspartic acid values). No insoluble collagen replicas could be obtained after either HCl or EDTA demineralization. Whole bone and gelatin isotope ratios varied greatly. In the case of SDM 16755, both the whole bone and gelatin extracts gave b1SN and bl3C values that fall within a range meaningful to interpretations of paleodiet. Attempting to assign a diet source to this skeletal material based on stable isotope determinations of noncollagenous residues is totally unjustified. In the case of the gelatin extracts, the stable isotope data is derived from amino acid residues (and contaminants) of unknown origin. However reasonable the isotopic values may seem, they may not be used for paleodiet interpretation. DISCUSSION

The stable carbon and nitrogen isotopes are not uniformly distributed throughout all the amino acids of bone collagen (Figs. I and 2). The variations seen in modern bone collagen are mirrored in the fossil collagen. This observation is in agreement with isotope values observed in the individual Table 6. Amino acid content in gelatin extracts ~oles of amino acids/ mg dry weight extract

Modern Fossil Fossil Fossil Fossil

fetal bovine whale (3 OAU 7) whale (M79-SB2) human (SOM 19254) human (SOM 16755)

6.0 6.2 6.2 1. 3 0.1

933

Table 7. Glycine/aspartic acid in whole bone, EDTA insoluble and gelatin residues Sample % Protein*

SDH 16755 SDH 19244 SDH 19254 Wha1e(3 DAU 7) 0.1

2.5

6.S

83.0

0.9 ne# 1.2

4.6 4. S 2.9

S.S 5.6 5.3

6.2 6.8 5.9

Glycine/aspartic acid Whole bone

EDTA Insoluble Gelatin

/lThere was no EDTA insoluble collagen replica for SDH 16755. *Amount of protein based on amino acid analysis relative to modern fetal calvaria which was assigned a value of 100%

amino acids of bovine Achilles tendon collagen and a fossil bison bone collagen (HARE and ESTEP, 1983). Glycine, threonine and serine values for both bl5N and bl3C vary significantly from both the mean values of the other amino acids and the total collagen stable isotope value. Because the loss of glycine, threonine and serine relative to the acidic amino acids has been reported (HARE, 1974, 1980), altered bl3C and b1SN values can be expected from some fossil bones such as SDM 19244. In theory, corrections could be made for small changes in the collagen amino acid profile and the resulting effect on the bl3C and b15N values. We estimate that for the human bone (SDM 19244), a glycine/aspartic value of 4.6 could mean that the b1SN value for the EDTA demineralized extract is as much as +2.0%0 too positive. Since blSN have been used to interpret the trophic levels of humans in the new world, it is important to determine the integrity of the collagen being analyzed (CHISHOLM et al., 1982; SCHOENINGER et al.. 1983; WALKER and DENIRO, 1986; DENIRO, 1987). Carbon/nitrogen values can not identify a small loss of glycine relative to aspartic acid. Although individual amino acid b13C and b1SN values are useful in correcting slightly altered collagen values, they should not be used for correcting or interpreting data derived from fossil bones that have a totally noncollagenous amino acid pattern. There are no data to suggest that the amino acids in bones such as SDM 16755, the wildebeest or zebra described in Table 5 are indigenous to the animal. The identification of the protein in fossil bones with a totally noncollagenous amino acid pattern remains one of the major challenges to the field. Preservation of several noncollagenous proteins has been demonstrated immunologically in both taphonomic and fossil bone material (TUROSS, 1987). While noncollagenous proteins do survive postmortem in some environments, the identity of the protein fragments in bones that have noncollagenous amino acid patterns has not been established. Little is known about the processes by which collagen molecules degrade to peptides that are removed from the bone, leaving aspartic (and glutamic) acid rich residues. Whether these acidic amino acids result from binding of collagen cleavage products to the mineral, the selective preservation of a noncollagenous component, or exogenous contamination is unknown. We have shown that in order to interpret the values produced for either carbon or nitrogen isotopes in collagen, a more detailed characterization of the analyzed material is necessary. Collagen in most fossil bones is not the same macromolecular entity that occurs in living specimens. While some cleavage of the collagen molecule into smaller fragments may not alter the biological isotopic signal, we present evidence that uneven loss of certain amino acids would perturb

934

N. Tuross, M. L. Fogel and P. E. Hare

an isotopic signal. Changes in fossil bone amino acid composition, whether they be diagenetic or analytic, that alter the glycine/aspartic acid valuefrom the biological, willperturb the stable carbon and nitrogen isotope values. These alterations in 015N and Ol3C that occur in fossil collagens with lowered glycine/asparticacid values will be large enough to affect dietaryinterpretation. However, the magnitude of these stable isotope alterations will not always take the observed 015N and Ol3C out of the range that is biologically possible. The collagen in fossil bones can exist in a variety of preservation states. These data indicate that the presence of a collagen replica, hydroxyproline and a glycine/aspartic acid value in the 6-7 range, will provide protein that retains the carbon and nitrogen isotope configuration of the living animal. Although previous investigatorshave indicated the usefulness of HCI and EDTA as demineralizing agents (ELDAOUSHY et al., 1978 and OLSSON et al., 1974), the fact that collagen remains as an insoluble replica after demineralization has been under-utilized in sample preparation for isotope analysis. The use of chelating agents such as EDTA can be used to give increased yields of insoluble collagen free of humic acid or EDTA contamination. In some cases, the traditional isolation procedures may furtherchangethe observedisotopicvalues. The lossofglycine relative to aspartic acid would occur if a gelatinization procedure of glycine-rich, collagen-derived peptides were solubilized in HCI and lost through the coarse filters generally used. The results on modem and well preserved fossil bones (Tables 2 and 3) indicate that collagen may be extracted by severalmethods to yieldvirtually identical stable isotope values for Ol3C and OI5N. In general, the removalof the inorganic phase of bone with EDTA resulted in greater yields of collagen.The use of mineral acids,coarsefiltering and extensive milling are all possible explanations for the relative loss of collagen observed with the HCI demineralization and the gelatinization protocols. The collagen yieldsin the moderatelyand poorly preserved bones (Tables 4 and 5) also were greatest for the EDTA extracted material. Collagen in most fossil bones exists as a partially degraded complex even when the amino acid composition of the fossil bone collagen is identical to purified collagen(TUROSS et aI., 1980). Gel electrophoresisindicates that collagen extracted under mild, dissociative demineralizingconditions from fossil bones has molecular weightfragments rangingfrom 4,000-100,000 daltons. Visualizationor quantitation of the organic component that ranges from free amino acids to 4,000 daltons has never been done; however, many fossil bones probably contain collagen degradation products in this range. Future comparisons of Ol3C and 015N for paleodiet analysiswill benefit from increased rigor in the characterization of the protein being analyzed.

CONCLUSIONS Well preserved fossil bone is a useful substrate for Ol3C and 015Nanalysis of collagen. At leasttwo extractionmethods can be used to isolate collagen from these bones. The lack of alteration in the chemical and isotopic compositions of

these well preserved bones agrees with NELSON et al. (1986) and DENIRO (1987). Fossil bones with less than twenty per cent of the original protein may not preserve the biological signal of collagen; they should be documented as to amino acid content, and screened as to the variability of the Ol3C and Ol5N values of more than one collagen extraction procedure. There is isotopic variation within the modem and fossil collagen molecule; glycine, serine and threonine have Ol3C and 015N values that differ from the other amino acids and from the aggregate collagen value. These isotopic variations are biochemical/metabolic effects, and are independent of possiblediageneticalterations. Loss of glycineis a likelydiagenetic trend in bone collagen, and its loss would result in enriched 015N and depleted Ol3C values relative to the true collagen isotopic signal. Acknowledgements-We wish tothank Thomas Hoering andThomas

Stafford for assistance with the individual amino acid separations andisotopic analysis, and Luis Cifuentes andThomas Hoering who provided critical reviews ofthe manuscript. This work was partially supported byNSF grant EAR-8313164 to M.L.F. and P.E.H. and a grant from the Wenner-Gren Foundation to N.T. Editorial handling: H. P. Schwarcz

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