www.elsevier.com/locate/chemgeo

Chemical Geology 22

Carbon and nitrogen isotopic fractionation in bone collagen

during chemical treatment

Masayo Minami *, Toshio Nakamura

Center for Chronological Research, Nagoya University, Nagoya 464-8602, Japan

Received 13 October 2004; received in revised form 15 June 2005; accepted 20 June 2005

Abstract

In measuring radiocarbon ages of fossil bone samples, it is necessary to extract pure bone protein from the samples by

chemical treatment. To evaluate the effect of the chemical treatment including XAD-2 chromatography on carbon and

nitrogen isotopic values, we measured C/N ratio, d13C and d15N in decalcified fraction, gelatin, hydrolysate, and XAD-

treated hydrolysate extracted from fossil bones together with modern samples. The C/N ratios of the collagen fractions

progressively decreased as chemical treatment proceeded. The d13C values of the fractions became more positive as

purification proceeded. There was an overall +0.5~+2.0x difference in d13C and a slight trend of decrease in d15N during

chemical processing and around +0.3x difference in d13C and +0.2x in d15N during XAD-2 treatment only. The large

change in C/N ratio and d13C of the hydrolysates following XAD-2 chromatography in the Bovine Achilles tendon

collagen standard could be explained by the removal of lipids unextracted before the XAD-2 treatment. There was no

difference in d13C between hydrolysates and XAD-treated hydrolysates in fossil bones because the latter contains

negligible preserved lipids. Fossil bones and lipid-extracted collagen standard showed the similar C /N change and isotopic

fractionation during sequential chemical treatment. Individual amino acid standards showed little to no increase in d13C

and d15N values during XAD-2 treatment, except for aspartic acid and glutamic acid, which showed pronounced increase

in d15N values. Furthermore, regarding amino acid compositions separated by XAD-2 treatment, alanine and glycine tend

to be enriched, while valine, threonine, isoleucine, leucine, and serine compositions tend to be depleted. The carbon and

nitrogen isotopic fractionation during sequential chemical treatment might reflect variation in the amino acid composition

of the extracted fractions due to degradation, such as decarboxylation, rather than removal of contaminants. The variation

during XAD-2 treatment is due to both degradation by HCl and isotopic fractionation related to the XAD-2 resin.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Fossil bone; Amino acids; Collagen; d13C; d15N; Isotopic fractionation

0009-2541/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.chemgeo.2005.06.005

* Corresponding author. Tel./fax: +81 52 789 3091.

E-mail address: minami@nendai.nagoya-u.ac.jp (M. Minami).

1. Introduction

In measurement of radiocarbon ages for fossil

bones, teeth, and tusks, the gelatin-extraction method,

2 (2005) 65–74

M. Minami, T. Nakamura / Chemical Geology 222 (2005) 65–7466

where gelatin is extracted from the acid/alkali-insolu-

ble residue of fossil samples by heating in acidic water

at 90 8C, has been commonly used to remove

adsorbed organic matter from bone protein (Longin,

1970; Arslanov and Svezhentsev, 1993). However,

alkali treatment can cause a considerable loss of

bone organic carbon, especially in poorly preserved

fossils, and the gelatin extraction without alkali-treat-

ment does not fully remove contaminants from bone.

In contrast, pretreatment of the fossils with the XAD-2

polymeric absorbent is effective for removing of for-

eign materials, such as humates and humic acids, to

obtain accurate dates (Stafford et al., 1987, 1988;

Minami and Nakamura, 2000; Minami et al., 2004).

The XAD-2 resin is considered the best material to

quantitatively extract inherent amino acids from fossil

bones, especially poorly preserved fossils.

However, when studying fossil bones, it is im-

portant to obtain accurate carbon and nitrogen stable

isotopic ratios together with accurate radiocarbon

ages. Obtaining carbon and nitrogen isotopic ratios

in collagen is a useful means of reconstructing the

diets of prehistoric mammals (e.g., van der Merwe

and Vogel, 1978; DeNiro and Epstein, 1980). For

this reconstruction, collagens extracted from bones

generally have been used, and for detailed recon-

struction, the carbon and nitrogen isotopic signa-

tures of individual amino acids in collagen are

needed as well as those of total collagen. Therefore,

the carbon and nitrogen isotopic fractionation during

chemical treatment including XAD-2 chromatogra-

phy of collagen from fossil bones should be deter-

mined. Stafford et al. (1988) reported that isotopic

fractionations due to XAD-2 chromatography are

negligible: +0.3x of d13C and +0.1x of d15N,

whereas Minami and Nakamura (2000) reported

that there is 1x enrichment in d13C and 0.1xdecrease in d15N by XAD-2 treatment for analysis

of collagen standards from bovine Achilles tendons.

The d13C values of the collagen standards also

became ~2x more positive during chemical treat-

ment other than XAD-2 chromatography. The iso-

topic variation by sequential extraction of collagen

standards probably originates from degradation of

the proteins, changes in the amino acid composition,

and/or decarboxylation rather than removal of or-

ganic contamination, because the collagen standards

are already purified reagents.

Isotopic fractionation of nitrogen and carbon is

considered to occur during peptide bond hydrolysis,

because the peptide bond containing 14N should rup-

ture preferentially (Stacey et al., 1952). Bada et al.

(1989) determined the relative d13C and d15N of free

amino acids, residual unhydrolyzed collagen, and

large protein fragments of bovine Achilles tendon

collagen. The residual collagen was ~20x enriched

in 15N, which was cited as evidence of isotopic frac-

tionation. However, the depletion of 13C in the resid-

ual collagen could not be explained.

Differentiation of carbon and nitrogen isotopes

during the hydrolysis process also was reported by

Silfer et al. (1992). They investigated kinetic isotope

effects associated with hydrolysis of the dipeptide

glycylglycine. The residual peptide became increas-

ingly enriched in 13C and 15N as the hydrolysis reac-

tion progressed, whereas free glycine became

increasingly depleted in 13C. The carbon isotopic

fractionation effect due to hydrolysis should be less

than that for nitrogen because carbon is present in

protein components other than the peptide bond,

whereas nitrogen is contained essentially entirely

within the peptide bond.

In the present study, we investigated carbon and

nitrogen isotopic fractionation in fossil bones during

sequential chemical treatment including XAD-2 chro-

matography. We compare these results with those

reported by Minami and Nakamura (2000) from col-

lagen standards. We also describe the isotopic frac-

tionation of individual amino acids and the changes in

the amino acid composition of collagen standards and

fossil bones after XAD-2 treatment.

2. Samples

The modern bones used in the experiment were

bovine Achilles tendon collagen standards (SigmaR

and NacalaiR) and a fragment of ivory. The amino

acid standards used were glycine (Gly), DL-alanine

(Ala), L-serine (Ser), L-valine (Val), L-tyrosine (Tyr),

L-proline (Pro), DL-asparatic acid (Asp), L-glutamic

acid (Glu), L-methionine (Met), and L-arginine (Arg)

(KishidaR). Animal fossil bone fragments of Japanese

deer (Cervus nippon), boar (Sus scrofa), snapping

turtle (Trionyx sinensis), and Japanese wolf (Canis

hodophilax) also were analyzed. The deer, boar, and

turtle fossil samples were collected from a shell

M. Minami, T. Nakamura / Chemical Geology 222 (2005) 65–74 67

mound excavated at Awazu submarine archeological

site at the southern basin of Lake Biwa, Shiga Pre-

fecture, Japan. The wolf sample was collected from a

cave at Mt. Kyonojo in Yatsushiro, Kumamoto Pre-

fecture, Japan.

3. Experimental

3.1. Sample preparation

The fossil bones and ivory sample were ultrasoni-

cated repeatedly in distilled water followed by 0.2 M

NaOH and rinsed with distilled water. The samples

were lyophilized and pulverized. These bone powders

and collagen standards were decalcified in 0.8 M HCl

for 24 h at 4 8C. The solution was centrifuged, and the

acid-insoluble residue was hydrolyzed with 6 M HCl

at 110 8C for 24 h. Solids were removed by centrifu-

gation, and then the filtered hydrolysate was treated

using XAD-2 chromatography. The XAD-2 chroma-

tography procedure was identical to that of Stafford et

al. (1988). The XAD-treated hydrolysate was eluted

with 6 M HCl and evaporated to dryness. Gelatin was

extracted from the acid-insoluble residue by heating in

acidic water at 90 8C. The gelatin solution was centri-

fuged and filtered before it was lyophilized.

The amino acid standards were each dissolved in 6

M HCl, and each of solutions was treated using XAD-

2 chromatography. The XAD-treated hydrolysates

were each eluted with 6 M HCl and evaporated to

dryness.

3.2. Stable isotope and radiocarbon analyses

The organic fractions obtained through the chem-

ical procedures described in the proceeding sections

were combusted at 850 8C in a sealed Vycor tube

together with CuO, Cu, and Ag wires. The N2 gas

produced was gathered onto molecular sieves after

removal of CO2 gas into liquid N2 traps in a vacuum

line and the CO2 gas was purified cryogenically.

Carbon and nitrogen isotope ratios were measured

by a Finnigan MAT-252 mass spectrometer. Precision

was 0.1x for d13C and 0.2x for d15N. A part of CO2

gas was reduced to obtain graphite by hydrogen, with

Fe catalyst, at 650 8C in a sealed Vycor tube. Mea-

surements of 14C / 12C ratios were performed by a

Tandetron AMS system at Nagoya University. Stan-

dards were graphite made from NBS oxalic acid

(SRM-4990C).

3.3. Derivatization procedure

The hydrolysate and XAD-treated hydrolysate

fractions of the collagen standard and some fossil

bones were derivatized to trifluoroacetic isopropyl

esters in a two-step procedure. The dried samples

were esterified in isopropanol and acetyl chloride for

1 h at 100 8C. Then the reaction was quenched, and

the amino acid isopropyl esters were evaporated to

dryness under a stream of N2 at 0 8C. The esters wereacylated for 10 min at 100 8C by addition of trifluor-

oacetic anhydride and dichloromethane. After acyla-

tion, the reagents were removed by evaporation under

a stream of N2 at 0 8C. The amino acid derivatives

were dissolved in dichloromethane prior to injection

for gas chromatography (Thermo Quest SSQ7000).

4. Results and discussion

The carbon and nitrogen isotopic ratios and C/N

ratio during sequential extraction of decalcified, hy-

drolyzed, and XAD-treated hydrolysate fractions from

fossil bones are shown in Table 1. The collagen frac-

tions (i.e., decalcified bone) yielded a C content of

about 40% to 50%, whereas the amino acid fractions

(i.e., hydrolysate and XAD-treated hydrolysate),

which were syrupy, gave a C content of about 30%

to 35%. The reason for the lower C content of the

amino acid fractions is that they contain considerable

amount of water. It also worth considering that some

of amino acids are destroyed and/or partially oxidized

during acid hydrolysis. The loss in hydrolysable

amino acids might partially account for the observed

decrease in carbon content.

4.1. C /N ratios

The C/N ratios of the collagen fractions progres-

sively decreased as chemical treatment proceeded

(Fig. 1). The decrease trend of C /N ratios was also

observed in previous studies (Minami and Nakamura,

2000; Minami et al., 2004). For the protein-derived

fractions, we obtained C/N ratios ranging from 2.6 to

3.6; these values are in agreement with the reported

value of 3.2F0.5 for collagen (Hare and von Endt,

Table 1

Variation of C /N ratio, d13C and d15N values during sequential chemical treatment of animal fossil bones and an ivory sample

Yield (%) C content (%) C/N ratio d13CPDB (x) d15NAIR (x) 14C age (BP)

Boar

Bone – 10.4 3.6 – – –

Decalcified bone 13.7 47.4 3.2 �21.0 +5.6 4410F80

Hydrolysate 3.2 34.8 3.0 �20.6 +4.7 –

XAD-treated hydrolysate – 30.0 2.9 �20.4 +4.9 –

Japanese deer

Bone – 8.2 4.0 – – –

Decalcified bone 7.0 43.7 3.0 �21.7 – 4300F90

Hydrolysate 2.3 36.3 2.8 �20.8 +4.2 –

XAD-treated hydrolysate – 30.0 2.7 �20.5 +4.4 4320F80

Snapping turtle

Bone – 6.8 4.2 – – –

Decalcified bone 5.5 43.0 3.2 �20.6 +9.3 4310F80

Hydrolysate 1.6 33.7 2.9 �19.8 – –

XAD-treated hydrolysate – 31.8 2.9 �19.4 +8.8 4600F100

Ivory

Bone – 16.6 3.0 �25.4 – –

Decalcified bone 31.5 50.2 3.1 �25.5 +11.2 Modern

Gelatin 21.5 44.6 2.8 �25.4 +11.3 Modern

XAD-treated hydrolysate – 35.5 2.8 �24.3 +11.5 –

Japanese wolf

Bone – 13.5 3.2 – – –

Decalcified bone 12.8 44.5 2.8 �18.9 +6.7 Modern

Gelatin 5.3 44.5 2.8 �18.7 +6.8 260F90

XAD-treated hydrolysate – 26.3 2.7 �17.9 +6.5 380F90

The error of 14C age is 1r.

M. Minami, T. Nakamura / Chemical Geology 222 (2005) 65–7468

1990). The C/N ratio is liable to depend upon the

stringency of the fossil sample preparation (Ambrose,

1990).

Decalcification of fossil bones can lead to a pro-

nounced decrease in the C/N ratio. This decrease is

partly due to removal of exogenous organic matter, for

which the C /N ratio can exceed 4.0 (Hedges and van

Klinken, 1992), and partly due to removal of bone

inorganic fraction. Bone inorganic fraction is a car-

bonate hydroxyapatite, which contains small percent-

age of CO3 that contributes to the total percentage of

bone carbon. Decalcification of bones, therefore,

should always lead to C /N ratio decrease. In Fig. 1,

modern samples, such as collagen standards and an

ivory sample, which might contain little exogenous

organic matter, showed smaller decrease of C/N ratio

than fossil bones on decalcification. The small C /N

ratio decrease for modern samples might be caused by

removal of bone inorganic fraction.

The decrease in the C /N ratios during hydrolysis

of decalcified fractions might be due to degradation of

proteins and/or decarboxylation by HCl rather than

removal of foreign organic matter, because the change

in the C /N ratios is also identical between the fossil

bones and the modern bones (Fig. 1). In addition, the

alteration by XAD-2 treatment might be attributed in

part to both degradation by HCl and fractionation by

the XAD-2 resin. However, the large decrease of C /N

ratios of the hydrolysates following XAD-2 chroma-

tography in the collagen standards is too large to be

explained by the above effects. The large decrease,

which is not observed in fossil collagens, seems

indicative of the removal of any other material than

amino acids. Lipid is thought to be a candidate as the

material. We tried to extract lipids from the SigmaR

Bovine Achilles tendon collagen using chloroform/

methanol (2 /1; v/v). The result is that the collagen

contains 15.2% lipids. Next, the lipid-extracted colla-

gen standard was decalcified, and the acid-insoluble

residue was hydrolyzed or gelatinized. The filtered

hydrolysate was treated using XAD-2 chromatogra-

phy, while the gelatin solution was centrifuged and

filtered. The results of C /N ratio, d13C and d15Nvalues for each fraction are shown in Table 2, together

2.5

3.0

3.5

4.0

4.5

Starting DB GC H XAD

C/N

rat

io

Fig. 1. Variation of C/N ratio during chemical treatment of bone

collagens. Abbreviations of fractions are as follows: DB: decalcified

bone fraction, GC: gelatin fraction extracted from a decalcified, H:

hydrolysate fraction of a decalcified bone, XAD: XAD-treated

hydrolysate fraction. Plot figures are shown as follows: closed

circle: boar (4400 BP), closed square: Japanese deer (4300 BP),

closed triangle: snapping turtle (4600 BP), open circle: ivory (mod-

ern), open square: Japanese wolf (380 BP), plus: NacalaiR collagen

standard (modern), cross: SigmaR collagen standard (modern), open

triangle: SigmaR collagen standard following extraction of lipids.

The data for the NacalaiR and non lipid-extracted SigmaR collagen

standards are from Minami and Nakamura (2000).

M. Minami, T. Nakamura / Chemical Geology 222 (2005) 65–74 69

with the result for the non lipid-extracted collagen

standard in Minami and Nakamura (2000).

The C/N ratios for lipid-extracted collagen frac-

tions were lower than those for non lipid-extracted

ones, and progressively decreased slightly as chemical

Table 2

C/N ratio, d13C and d15N values for non lipid-extracted and lipid-extra

extracted from the collagen

C content (%) N content (%

Non lipid-extracted1)

Collagen 50.6 13.0

Decalcified fraction 49.1 12.9

Hydrolysate 24.3 6.8

XAD-treated hydrolysate 25.8 8.1

Lipid-extracted

Collagen 44.3 15.8

Decalcified fraction 41.0 14.1

Gelatin 41.5 14.8

XAD-treated hydrolysate 27.4 9.4

Lipid 63.0 b0.1

1)The data for the non lipid-extracted SigmaR collagen standards are fro

2)The ratio cannot be estimated because the nitrogen content in lipids can

treatment proceeded, similar to those for non lipid-

extracted ones (Fig. 1). Lipids, comprised mainly of

triacylglycerols, with a lower abundance of diacylgly-

cerols and free fatty acids, are carbon-rich and negli-

gible nitrogen. The lower C /N ratios for lipid-

extracted collagen fractions might be caused by the

removal of lipids. The large decrease in C /N ratio of

hydrolysates following XAD-2 chromatography in

non lipid-extracted collagen also seems indicative of

the removal of lipids. The XAD-2 resin might remove

unextracted lipids from hydrolysate fractions of mod-

ern samples.

4.2. Carbon and nitrogen isotopic ratios

The d13C values of the fractions extracted from

fossil bones increased as chemical treatment pro-

ceeded (Fig. 2). The increase trend also is observed

in previous studies (Minami and Nakamura, 2000;

Minami et al., 2004). There was an approximate

overall 0.5x to 2.0x variation in d13C between

starting and XAD-treated hydrolysate fractions. The

fossil bones showed the similar extent of fractionation

in d13C values as did the modern bones, though d13C

of the hydrolysate for modern samples increased sud-

denly by XAD-2 chromatography. The carbon isoto-

pic fractionation between hydrolysate and XAD-

treated hydrolysate of the fossil bones was around

+0.3x, similar to the result of Stafford et al. (1988)

but different from that of the modern samples. The

carbon isotopic fractionation by XAD-2 treatment

cted SigmaR Bovine Achilles tendon collagen standards, and lipid

) C/N ratio d13CPDB (x) d15NAIR (x)

3.9 �15.1 +7.3

3.8 �14.6 +7.3

3.6 �14.1 +7.2

3.2 �13.0 +7.2

2.8 �13.9 +7.4

2.9 �12.8 +7.1

2.8 �13.0 –

2.9 �12.9 +7.1

–2) �19.6 –

m Minami and Nakamura (2000).

not be measured.

-26

-24

-22

-20

-18

-16

-14

-12

Starting DB GC H XAD

δ 13

CP

DB

(‰)

Fig. 2. Carbon isotopic fractionation during chemical treatment of

bones. Abbreviations and plot figures are the same as for Fig. 1.

Added plot figures: open square: Uwa sample (44000 BP), closed

square: Mawaki sample (5300 BP). The data for the NacalaiR and

non lipid-extracted SigmaR collagen standards are from Minami and

Nakamura (2000), and those for the Uwa (molar fossil of Nau-

mann’s elephant collected from the bottom of Uwa-sea, Ehime,

Japan) and Mawaki samples (dolphin fossil excavated from the

Mawaki archeological site, Ishikawa, Japan) from Minami et al.

(2004).

2.0

4.0

6.0

8.0

10

12

Starting DB GC H XAD

δ 15

NA

IR (‰

)

Fig. 3. Nitrogen isotopic fractionation during chemical treatment of

bones. Abbreviations and plot figures are the same as for Fig. 2. The

data for the NacalaiR and non lipid-extracted SigmaR collagen

standards are from Minami and Nakamura (2000), and those for

the Uwa sample are from Minami et al. (2004).

M. Minami, T. Nakamura / Chemical Geology 222 (2005) 65–7470

might have been not only due to kinetic fractionation

by chromatography, but also due to removal of exog-

enous organic matter, degradation of proteins and/or

decarboxylation by HCl. Considering that the enrich-

ment in d13C during sequential chemical treatments

including XAD-2 treatment is gradual, the carbon

isotopic fractionation during XAD-2 treatment could

be due to degradation of proteins rather than kinetic

fractionation by chromatography.

The reason why modern samples showed the larger

increase in d13C value by XAD treatment is that any

other material than amino acids, such as lipid, could

remain in the gelatin and hydrolysate fractions of mod-

ern samples. The d13C values for decalcified, gelatin

and XAD-treated hydrolysate fractions in lipid-

extracted collagen were fixed to about�12.9x, higher

than the decalcified and gelatin fractions for no-pre-

treated collagen, and similar to its XAD-treated hydro-

lysate fraction (Fig. 2). Since the d13C of lipids

extracted from collagen was analyzed to be �19.5x,

more 13C-depleted than collagen proteins, the higher

value of�12.8xmight be caused by removal of lipids.

It is noteworthy that the d13C values for non lipid-

extracted and lipid-extracted collagens are almost the

same for XAD-treated hydrolysate fraction. This sug-

gests that XAD-2 treatment removes lipids from mod-

ern bone collagen. On the other hand, there was no

difference between hydrolysates and XAD-treated

hydrolysates in fossil bones because the latter contains

negligible preserved lipids, compared to modern ones.

Therefore, the decrease of C /N ratio and carbon isoto-

pic fractionation during chemical treatment in modern

samples includes the change by removal of lipids from

bone proteins.

The d15N values decreased slightly during sequen-

tial chemical processing (Fig. 3), but the values

tended to be little positive by XAD-2 treatment.

The nitrogen isotopic fractionation by XAD-2 chro-

matography for collagen standards of bovine Achilles

tendons was reported to be +0.1x by Stafford et al.

(1988) and �0.1x by Minami and Nakamura (2000).

From these results, the nitrogen isotopic fractionation

during XAD-2 treatment for collagens either is neg-

ligible or shows slight enrichment. The d15N values

for decalcified and XAD-treated hydrolysate fractions

in lipid-extracted collagen were ~+7.2, similar to the

value for non lipid-extracted collagen. Lipids contain

little nitrogen, and extraction of lipids from collagen

has no effect on its d15N. Therefore, there might be

no difference in nitrogen isotopic fractionation bet-

ween fossil bones and modern samples. We should

M. Minami, T. Nakamura / Chemical Geology 222 (2005) 65–74 71

consider here that the d15N values during chemical

treatments do not always show a decrease, and that

those of hydrolysate and XAD-treated hydrolysate

show a variation with the same order of analytical

error. More statistically sufficient measurements of

d15N are needed to discuss further on the d15N var-

iation during chemical treatment.

Hydrolysis enriches residual, unhydrolyzed protein

in 15N whereas 13C typically is relatively unaffected

(Bada et al., 1989). Therefore, chemical treatment

with HCl should deplete acid-soluble, hydrolyzed

fractions of 15N. The d15N decrease of the hydrolyzed

fractions that our results indicated in Fig. 3 might

have been the result of peptide bond hydrolysis. How-

ever, 13C, which typically is relatively unaffected by

hydrolysis, was enriched during hydrolysis in the

present study. When decarboxylation occurs in a frac-

tion, 12C is removed preferentially relative to 13C, and

the residual fraction becomes enriched in 13C. The

same enrichment in d13C among modern collagen

standards and samples, all of which contained scarcely

any foreign organic matter, and the fossil bones,

which contained contaminants, indicates that isotopic

fractionation during sequential chemical treatment

might depend on degradation-induced changes in the

amino acid composition of the fractions rather than on

removal of contaminating material.

4.3. XAD-treatment-induced changes in carbon and

nitrogen isotopic ratios of amino acid standards

Hydrolysate fractions contain numerous amino

acids, and the carbon and nitrogen isotopic fraction-

Table 3

Comparison of d13C and d15N values in starting reagents and XAD-treate

Starting amino acids

d13CPDB (x) d15NAIR

Glycine �33.8 +0.4

DL-a-Alanine �25.3 �3.0

L-Valine �20.2 �5.5

DL-Asparatic acid �22.8 �4.8

L-Glutamic acid �13.0 �12.3

L-Arginine �13.8 �7.6

L-Serine �37.1 +1.8

L-Proline �12.3 +10.7

L-Methionine �32.4 +1.4

L-Tyrosine �17.0 +5.8

Figures in parenthesis show variation in d13 CPDB and d15 NAIR by XAD

ation of hydrolysates is the sum of the fractionations of

the individual amino acids. Therefore, we investigated

the carbon and nitrogen isotopic fractionation of some

amino acid standards during XAD-2 treatment (Table

3). The slight d13C increase for these individual amino

acids might bring enrichment in 13C for the fractions of

all amino acid combined, i.e., the XAD-treated bulk

hydrolysates. Hydroxyproline is a relatively abundant

amino acid in collagen, and it could effect isotopic

fractionation of bulk hydrolysates. However, consid-

ering that carbon isotopic fractionation during XAD-2

treatment of the bulk hydrolysates in fossil bones is not

large so much, around +0.3x, and that the fraction-

ation for individual amino acid is 0~+0.1x, hydroxy-

proline would show little isotopic fractionation by

XAD-2 treatment.

The d15N values also increased during XAD-2

treatment: +0.1x for Ser, Val, and Pro and approxi-

mately +0.5x to 0.6x for Asp and Glu. The XAD-2

resin can separate polar amino acids from the less-

polar humates. Because Asp and Glu each contain two

carboxyl radicals, they might behave differently from

the other amino acids. Silfer et al. (1994) reported

that, compared with neutral amino acids, acidic amino

acids appear to be more susceptible to kinetic frac-

tionation from preferential hydrolysis or other diage-

netic reactions. The pronounced fractionation of d15Nthat we observed for these two amino acids might

have been caused by preferential kinetic fractionation

or by altered absorbability onto the XAD-2 resin

because of their increased polarity.

Considering the changes in carbon and nitrogen

isotopic ratios of amino acid standards by XAD-2

d fractions for individual amino acid standards

XAD-2-treated amino acids

(x) d13CPDB (x) d15NAIR (x)

�33.8 (0.0) +0.4 (0.0)

�25.2 (+0.1) �3.0 (0.0)

�20.2 (0.0) �5.4 (+0.1)

�22.7 (+0.1) �4.2 (+0.6)

�13.0 (0.0) �11.8 (+0.5)

�13.7 (+0.1) �7.6 (0.0)

�37.1 (0.0) +1.9 (+0.1)

�12.3 (0.0) +10.8 (+0.1)

�32.4 (+0.1) +1.4 (0.0)

�17.0 (0.0) –

-2 treatment.

M. Minami, T. Nakamura / Chemical Geology 222 (2005) 65–7472

treatment, together with those of C /N ratios, d13C,

and d15N, the degradation by HCl occurred in a

XAD-treated fraction, and 12C and 14N were re-

moved preferentially relative to 13C and 15N, with

the result of slight enrichment in d13C and d15N.

By the other chemical treatments as well as XAD-2

treatment to collagen, the degradation by HCl also

occurs in individual amino acids, which compose a

collagen fraction, and thus the d13C and d15N

values of the fractions increase as chemical treat-

ment proceeds. The d15N decrease of the hydroly-

sate fractions might be caused by isotopic fractionation

by preferential peptide bond hydrolysis rather than

degradation.

4.4. Amino acid compositions of hydrolysates and

XAD-treated hydrolysates

The amino acid compositions in the hydrolysates

and XAD-treated hydrolysates from the two collagen

standards and selected fossil bone samples are shown

in Table 4. The hydrolysates contained high concen-

trations of Gly, Ala, Pro, and Glu. Collagen generally

has a characteristic amino acid composition, contain-

ing unusually high abundance of Gly, Ala, Pro, and

hydroxyproline (Hedges and van Klinken, 1992), and

our results coincide with these attributes of collagen.

Furthermore, the ratios of Asp /Gly and Asp /Pro

Table 4

Amino acid compositions (Amol/mg) of hydrolyzed and XAD-treated hyd

some fossil bones

Collagen standard

NacalaiR SigmaR Boar

H XAD H XAD H

Alanine 0.70 1.29 0.55 0.46 0.93

Glycine 2.18 3.47 1.57 1.77 2.13

Valine 0.09 0.11 0.80 0.31 1.09

Threonine 0.11 n.d. 0.46 0.34 0.37

Isoleucine n.d. n.d. 0.51 n.d. 0.38

Leucine 0.11 n.d. 0.19 n.d. 0.17

Serine 0.27 0.11 0.53 0.36 0.28

Proline 0.49 0.86 0.23 0.43 0.35

Aspartic acid 0.12 0.20 0.22 0.34 0.20

Glutamic acid 0.45 0.37 0.38 0.59 0.37

Asp/Gly 0.06 0.06 0.14 0.19 0.09

Asp/Pro 0.24 0.23 0.96 0.79 0.57

H: hydrolysate, XAD: XAD-treated hydrolysate, n.d.: not determined.

show collagen-like amino acid composition, because

Gly and Pro are usually abundant in collagen, whereas

Asp is abundant both in bone non-collagenous protein

and in most protein (e.g., DeNiro and Weiner, 1988;

Hedges and van Klinken, 1992). The result shows that

the analyzed fossil samples are well-preserved, while

it is strange that the ratios for modern SigmaR colla-

gen are higher than those for fossil bones. The batch

of the collagen contains ~15% lipids by dry weight,

far from being pure protein as mentioned above. The

higher ratios of Asp /Gly and Asp /Pro, therefore,

might be derived by contaminant contained in the

modern collagen.

The individual amino acid concentrations after

XAD-2 treatment tended to be increased for Ala,

Gly, and Pro, but decreased for Val, threonine (Thr),

isoleucine (Ile), leucine (Leu), and Ser (Fig. 4). The

differential depletion of amino acids might be caused

by diagenesis of hydrolysate fractions and/or purifi-

cation of collagenous amino acids by XAD-2 treat-

ment. Since the d13C and d15N of individual amino

acids hardly change by XAD-2 treatment, Ala, Gly

and Pro might be enriched by decomposition of the

other amino acids. The individual amino acids in a

hydrolyzed fraction each have different d13C and

d15N values; thus, changing the amino acid composi-

tion of the fraction changes its overall d13C and d15N

values. It is not decisive yet whether this is due to

rolyzed fractions for bovine Achilles tendon collagen standards and

Fossil bone

Deer Turtle Wolf

XAD H XAD H XAD XAD

1.03 0.71 1.00 1.06 1.27 1.39

2.20 1.83 2.40 2.47 3.13 4.26

1.06 0.85 0.90 0.73 0.17 0.15

0.12 0.35 0.21 0.26 0.14 0.20

0.12 0.36 0.09 0.43 0.04 n.d.

n.d. 0.14 n.d. 0.21 0.08 n.d.

0.13 0.26 0.13 0.21 0.17 0.36

0.25 0.37 0.36 0.42 0.73 1.06

0.16 0.26 0.23 0.21 0.35 0.44

0.39 0.55 0.43 0.35 0.11 0.24

0.07 0.14 0.10 0.09 0.11 0.10

0.64 0.70 0.64 0.50 0.48 0.42

-1.0

-0.5

0.0

0.5

1.0

1.5

Ala Gly Val Thr Ile Leu Ser Pro Asp Glu

BoarDeerTurtleNacalai® collagenSigma® collagen

Dev

iatio

n af

ter

XA

D-2

trea

tmen

t

(µmol / mg)

Fig. 4. Change in concentrations of individual amino acids by

XAD-2 treatment of hydrolyzed fractions in the collagen standards

and some fossil bones.

M. Minami, T. Nakamura / Chemical Geology 222 (2005) 65–74 73

differences between modern and fossil samples or is

species-specific.

5. Conclusions

Carbon isotopic fractionation and C/N-ratio

change took place during chemical extraction of

bone collagens and amino acids. The fractionation

likely result in isotopic enrichment of the heavier

carbon and nitrogen atoms because of degradation,

decarboxylation and variation in the amino acid com-

position of the extracted fractions, in addition to the

removal of contaminating matter. The 15N depletion

during hydrolysis might be caused by isotopic frac-

tionation by more preferential peptide bond hydrolysis

than degradation. The large changes in C/N ratio and

d13C in SigmaR Bovine Achilles tendon collagen

standard might be caused by the presence of lipids,

which are carbon rich and contain negligible nitrogen.

The XAD-2 resin could remove unextracted lipids

from hydrolysate fractions of modern samples.

The variation by XAD-2 treatment is due to both

degradation by HCl and some minor isotopic fraction-

ation related to the XAD-2 resin. This effect of frac-

tionation is increasingly important as the degree of

chemical treatment increases. Therefore, isotopic anal-

ysis of amino acid fractions extracted from archaeo-

logical bones can yield misleading isotopic ratios.

These, in turn, complicate the use of such isotopic

results in palaeodietary reconstructions. More studies

of amino acids, peptides, and proteins are required to

establish the isotopic fractionations of nitrogen and

carbon. In addition, analysis of other fossil bones in

which the quality of preservation differs, such as

poorly preserved fossil bones, is needed to systemat-

ically study isotopic fractionation during chemical

treatment of bones. These results will help clarify

the extent to which isotopic fractionation during

chemical treatment obscures the original isotopic

composition of the amino acids in fossil bones.

Acknowledgements

We are grateful to Mr. I. Iba (the Board of Educa-

tion of Shiga Prefecture) and Mr. N. Kitamura (Kuma-

moto Museum) for providing the fossil bone samples.

We thank Prof. K. Ohta (Nagoya University) for an-

alyzing the amino acid composition of the bone, and

Ms. A. Ikeda (Nagoya University) for providing the

ivory sample and some data. The authors are grateful

to anonymous reviewers for useful comments. This

work was supported in a part by a Grant-in Aid for

Scientific Research on Priority Areas from the Minis-

try of Education, Culture, Sports Science and Tech-

nology (MEXT) of Japan (subject #15068206). [PD]

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