ANNALES HISTORICO-NATURALES MUSEI NATIONALIS HUNGARICI Volume 93 Budapest, 2001 pp. 239-257.

Introduction of amino acid racemisation based age estimation into paleoanthropological research

J . C S A P Ó ' - , Z S . B E R N É R T - , Z S . C S A P Ó 1 , G. P O H N 1 , Zs. C S A P Ó - K I S S 1

L . K Ö L T Ő ' , I . S Z I K O S S Y 2 & S. N É M E T H Y '

'University of Kaposvár, Faculty of Animal Sciences, Department of Biochemistry and Foodstuff Chemistry, H-7400 Kaposvár, Guba S. u. 40, Hungary. E-mail: csapo@mcdl.atk.u-kaposvar.hu

Hungarian Natural History Museum, Department of Anthropology H-1082 Budapest, Ludovika tér 2, Hungary. E-mail: bernert@ant.nhmus.hu

' University of Pécs, Faculty of Jurisprudence, H-7622 Pécs, 48-as tér 1, Hungary E-mail: csapozs@pte.ajk.hu

Directorate of Somogy County Museums, H-7400 Kaposvár, Fő u. 10, Hungary E-mail: Kol to @ smmi. hu

Ostrami Limited, 66 Cleveland Avenue, Darlington, Co. Durham, DL3 7HG, Great Britain

Csapó , J., Bernér t , Zs., Csapó , Zs., Pohn, G., Csapó-Kiss , Zs., Köl tő , L . , Szikossy, I . & N é m e t h y , S. (2001): Introduction of amino acid racemisation based age estimation into paleoanthropological re­search. - Annales historico-naturales Musei nationalis hungarici 93: 239-257.

Abs t r ac t - This study describes the application and results of an age estimation method so far not in ­troduced into the inventory o f historical anthropology. The authors analysed the teeth o f 126 ind iv id­uals (age range 17-86 years) and found a high degree o f correlation between D-aspartic acid content, D / L rate o f aspartic acid and the individual age o f specimens. They developed a method for age deter­mination based on D-aspartic acid content and on the racemisation of L-aspartic acid o f teeth. D-glutamic acid, beside aspartic acid, was found to be eminently suitable for the estimation of ind i ­vidual age, as it showed a sufficiently high sensitivity. Calibration curves based on these investiga­tions were used for the age estimation o f 65 adults (39 males and 26 females) o f unknown individual age from the Avar Period series of Ke rek i -Homokbánya (Hungary). The age distribution o f the sam­ple was the fo l lowing: 39 individuals (60%) belonged to the adult age group, 22 persons (34%) to the mature and 4 (6%) to the senile one. The correlation between our results and those obtained using standard paleoanthropological methods was over 0.9. Wi th 3 figures and 5 tables.

Key words - Amino acid racemisation, D-aspartic acid, D-glutamic acid, age estimation, dental method, physical anthropology, Avar Period.

Correspondence to JÁNOS CSAPÓ and to ZSOLT BERNÉRT.

INTRODUCTION

The possibly most accurate estimation of age at the time of death is a signifi­cant part of thé study of historical populations. In this paper we report the results achieved by an age estimation method that so far was not utilised for the anthropo­logical analysis of historical populations.

In everyday practice age means the timespan between the birth and death of any individual human being. It is traditionally measured in years, months, weeks, etc. In other words it is calendar or chronological age. The usual age estimation methods of historical anthropology observe and analyse the changes occurring on the skeleton and on the teeth with the progress ot age. Growth, maturation and ag­ing are all processes that leave their marked, characteristic traces on human bones and teeth, therefore the estimation of the so called skeletal and dental ages could be founded on these. Skeletal and dental ages come from the biological age of any given individual. I f and when the person's biological age is close to his or her cal­endar age our estimate for his or her skeletal (or dental) age can fall very close to his or her calendar age ( U B E L A K E R 1989). This way we are able to produce an indi­rect estimation of calendar age via biological age determination.

When estimating age the anthropologist analyses ontogenesis, the biological aging of the human organism. These processes are determined by a multitude of external (environmental) and internal (genetic) factors. Chronological age runs at a steady pace, but the passing of biological age can be and really is very varied be­tween individuals and even within one human frame too, among its constituent parts. This is the reason for which we cannot achieve a truly accurate calibration of our age measuring methods compared to the passing of chronological time: this sort of calibration could be nothing else but an individual one. As biological and calendar ages often do not concur, chronological age in paleoanthropology may be estimated only within certain intervals (5yrs at least). There can be no other realis­tic aim for us but to score within these limits with the greatest plausibility possible.

Some constituents of the human body, such as teeth, are easy to examine even centuries after the death of the individual, as their changes are not significantly af­fected by the environment. Their analysis could significantly improve the accuracy of age estimation.

A REVIEW OF A M I N O ACID R A C E M I S A T I O N RESEARCH

H E L F M A N & B A D A (1975) were the first to declare that the aspartic acid racemisation process within teeth could be utilised to estimate the age of l iving ani-

mais and of humans. Then they established the reaction speed coefficient of aspar­tic acid for human teeth at 8,29x10"* year"1, but they measured it at 7 , 8 7 x l 0 - 4

year 1 a year later ( H E L F M A N & B A D A 1976). B A D A & B R O W N (1980) produced a calibrating diagram by plotting In (1+D/L)/(1-D/L) against time. They found a sat­isfactory level of conformity between the actual age data and the data estimated on the basis of amino-acid racemisation.

G l L L A R D et al. (1990) analysed the D-Asp content of molars and premolars. They failed to establish significant differences between the D-Asp contents of the same teeth when taking samples from several parts of their crowns and roots. O H T A N I & Y A M A M O T O (1992) found significant differences between estimated and actual ages when comparing the D-Asp contents of dental enamel and dentin substance. They detected that racemisation ( k A s p : 5.75x10"4 year"') is much faster in dentin than in enamel ( k A s p : 4.47x 10^ year"1). They concluded that age could be more accurately estimated on the basis of dentin (±3 years) than on the basis of enamel (±2 -11 years). They also calculated post-mortal reaction speed coeffi­cients for an average temperature of 15 °C (dentin: k A : 9.70x10" 8 year"1 and enamel: k A s p : 1.330x10"7 year 1 ) .

RlTZ et al. (1993) analysed the dentin substance of third molars' roots and they arrived at the conclusion that racemisation of the root's dentin was somewhat different to that of the crown's dentin. They produced a special calibration diagram for those cases where age has to be determined but the crown substance was dam­aged or impaired. They established that the degree of Asp racemisation was mult i­plied in acid solvent proteins compared to non-acid solvent ones.

O H T A N I (1994) examined Asp racemisation on central and lateral incisors, on first and second molars and he also treated the averages of these. He found a close correlation of actual ages and D/L Asp ratios. O H T A N I concluded that racemisation within deciduous teeth was an excellent indicator for the estimation of individual age, but racemisation of permanent was far less useful for the same purpose. R l T Z et al. (1995) worked out a biopsical sampling technique for teeth. Thanks to the strictly regulated nature of the sampling process ages estimated on the basis of Asp racemisation presented a very close conformity to actual ages. R l T Z et al. (1995) established a margin of error not larger than ±year for 46.4% of the cases analysed and the error of age estimation never went beyond the ±5 years l imit .

Recently we could see a dramatic progress of analytic methods but still there is no process that could perfectly separate the enantiomers of all protein constitu­tive amino acids in a single step. D-Asp was easy to trace and to separate from L-Asp and from other amino acids and the enantiomers of those, and these charac­teristics made it evidently suitable tools for analytic methods.

The aforementioned publications did not even bring up the possibility of the utilisation of any other amino acid but Asp for individual age estimation. C S A P Ó et al. (1994, 1998) established the racemisation half-periods of amino acids. They found the racemisation half-period of Asp was 13,500 years, that of histidine (His) 5500 years, the half-period of phenylalanine (Phe) was 8500 years, that of tyrosine (Tyr) was 13,500 years. Serine (Ser) had a value of 16,500 years, threonine (Thr) had a half-period of 17,000 years, while Glu bore one of 28,500 years. Alanine (Ala) had a racemisation half-period of 32,000 years, isoleucine (He) had one of 110,000 years, leucine (Leu) had one of 140,000 years and valine (Val) 180,000. Consequently D-enantiomers of amino acids with faster racemisation than that of Asp (namely His, Phe, Tyr) promise to be just as good indicators of individual age as Asp is. The two amino acids falling between Asp and Glu (Ser, Thr) could also provide useful information on age.

A R C H A E O L O G I C A L DESCRIPTION OF THE POPULATION A N A L Y S E D

Kereki is a village to the south of Lake Balaton and to the south of Kőröshegy as well. There in a sandpit an Avar cemetery came to light there and its rescue ex­cavation uncovered 151 graves in 1987-1988 ( K Ö L T Ő 1988, 1991). Four groups of graves constituted this untouched cemetery of a late Avar community. The people buried there bore markedly archaic features. Burials took place for 3^4 generations probably from the first third of the 8 t h Century. A peculiarity of this cemetery was that the unusually undisturbed (for this period) graves contained 21 belt-garnitures.

"CLASSIC" A G E E S T I M A T I O N METHODS OF ANTHROPOLOGY

The possible most accurate estimation of age at the time of death was consid­ered especially important for the paleodemografical and paleoostomatological analysis of the historical population of Kereki-Homokbánya ( B E R N É R T 1996«, S Z I K O S S Y & B E R N É R T 1996), therefore we applied a quite large number of inter­nationally accepted age estimation methods.

On adults we examined the changes of the sternal ends of ribs ( I S C A N et al. 1984, 1985), the surface alterations of os pubis fades symphyseosa ( T O D D , cit.

U B E L A K E R 1989), and the degree of ectocranial ( M E I N D L & L O V E J O Y 1985,

V A L L O I S , cit. F A R K A S 1 9 7 2 ) and endocranial ( N E M E S K É R I et al. 1 9 6 0 ) ossifica­tion of the brain case's suturae. We also scored the progress of organic matter's de-mineralisation in the roots of permanent teeth ( L A M E N D I N et al. 1 9 9 2 ) . To simplify the necessary calculations we used tables developed by ourselves ( B E R N É R T 1996b). To determine age we observed the wear of permanent teeth ( P E R I Z O N I U S , cit. E R Y

1 9 9 2 ) when its cause could be traced back to some sort of physiological process. Whenever the preservation of the skeleton permitted it we attempted to esti­

mate age within a five years envelope.

DISTRIBUTION OF THE HISTORICAL POPULATION SAMPLE BY SEX A N D AGE

We drew into this present analysis 6 5 adult members ( 3 9 men, 2 6 women) of this historical population (Tables 1 and 3 ) . Table 1 provided the distribution by sexes and by age groups as proposed by M A R T I N ( 1 9 2 8 ) . The adultus age group

Tab le 1. Distribution according to sex and age groups o f the individuals

Age groups Male Female Together

Adultus 20-24 0 1 1

25-29 2 2 A

30-34 9 5 14

35-39 8 10 IS

Total 20-39 19 18 37

Maturus 40^14 6 1 7

45^19 5 2 7

50-54 3 1 4

55-59 1 2 3

Total 40-59 15 6 21

Senium 60-64 0 0 0

65-69 2 1 1

70-74 1 0 1

75-79 0 0 0

Total 60-79 3 1 4

Total 20-79 37 25 62

was represented by 39 individuals from this 65 (60%), the maturus group by 22 persons (34%), the senium by 4 people (6%). Less than 5% of this historical popu­lation survived to their 60th years (BERNÉRT 1996b), and they lost almost all their teeth for this old age. This was the reason why the age group senium was repre­sented by such a low percentage within the sample.

There were two men (one of 25-34 years, one of 40-49 years) and one woman whose age could be estimated only within a 10 years interval because of the poor preservation of their remains.

Not all the selected age determination methods could be applied for all indi­viduals because of the incomplete preservation of the skeletons (Table 2). Natu­rally at least partial preservation of the sets of teeth was a prerequisite of dental enamel sampling. Root transparency and wear of teeth could be observed on al­most all persons examined. Bad preservation of the skeletons made the application of other age estimation methods possible only in a smaller number of cases.

Preparation ofteeth - T h e protein content of dental dissections was scored by Kjel-Foss rapid nitrogen analyser. The amino acid composition of teeth was mea­sured Labor M I M amino acid analyser, D- and L-amino acid contents were mea­sured by Hitachi Merck LaChrom high performance liquid Chromatograph. Pro­teins and all amino acids were identified by routine procedure according to ac­cepted standards. D- and L-amino acid enantiomers were separated and identified by the following method:

Pyrex reusable hydrolysis tubes were used for the hydrolysis of proteins. In the process of protein hydrolysis we filled 20-50 mg of compound into the Pyrex hydrolysis tubes that were washed with hydrochloric acid and twice distilled water before. 8 cm 3 of 6 M hydrochloric acid was added to each sample and nitrogen was bubbled through the system by a glass capillary tube. Hydrolysis tubes were imme­diately sealed after nitrogen bubbling, and then hydrolysis was carried out with 6 M hydrochloric acid on 110 C° for 24 hours, on 160 C° for 30 and on 170 C° for 45 minutes. More than one methods of hydrolysis were necessary because we wanted to determine which one of them produced the smallest level of racemisation. I t was an important consideration as D-amino acids could also be produced as by-products in the process of racemisation and this could significantly modify the accuracy of calibration. Our earlier experiments (CSAPÓ et al. 1997) indicated that protein hydrolysis carried out on a higher temperature but for a shorter time pro­duced a lower level of racemisation. Therefore we chose this way for our present series of experiments. Tubes were cooled to room temperature after completing hydrolysis, hydrochloric acid was removed by liophylisation, and the remaining substance was dissolved in 0.01 M hydrochloric acid. Finally, the hydrolysatum was filtered and kept on -25 °C until the titration of D and L amino acids.

Table 2 . Methods for age estimation o f adults

Methods for age estimation

Transparency o f root and periodontosis

Degree o f abrasio o f the teeth

Ectocranial ossification o f sutures

Endocranial ossification o f sutures

Changes o f surface o f the faciès symphyseos ossis pubis

Changes o f surface of sternal extremity of the rib

IDENTIFICATION OF D - A N D L-ENANTIOMERS

Apparatus - D- and L-amino acids were identified by a high efficiency

LaChrom Hitachi Merck liquid Chromatograph. The apparatus was built up from a

LaChrom D-7000 System Manager device, a LaChrom L-7100 HPLC pump, a

LaChrom L-7300 column thermostat, a LaChrom L-7200 Autosampler, a

LaChrom L-7400 programmable U V detector and a LaChrom 7480 fluorescent

detector. Amino acid enantiomers were separated after o-phtaldialdehyd (OPA)

and 2,3,4,6-tetra-O-acetil-l -t io-ß-D-glycopyranosid (TATG) derivatisation in a

non-chiral column aided by gradient elution.

Chemicals - Acetonitril and methanol were purchased from Rathburn

(Walkerburn, U.K.) , amino acid standards, OPA and T A T G were purchased from

Sigma (St. Louis, Mo.) . Elution puffers were produced from mono- and

dinatrium-hydrogen-phosphat.

Derivatisation - Reaction was executed in 120 urn micro phials put into 1.8

cm 3 volume ampoule with teflon covered internal covers and caps. The automatic

sample feeder was programmed to mix the sample (free amino acids or protein

hydrolysate distilled in nitrogen stream) dissolved in 90 ml of boric puffer (0.4M;

pH:9.5) with 15 ml of reagent (8 mg of OPA and 44 mg of T A T G dissolved in 1

cm 3 of methanol). After this the solution was thoroughly mixed by repeated suck­

ing up and releasing back, then it was left to rest for 6 minutes. The injecting device

was carefully washed through and then we injected 25 mm of this reaction com­

pound into the column of analyser. When finishing injection the system was three

times flushed through by a 70:30 ratio 100 ml acetone water mixture.

Separation and determination of enantiomers - Enantiomers were separated

by reversed phase chromatography according to the method of E l N A R S S O N et al.

(1987). We used a Kromasil octil (C8) loaded analytical column of 25x4.6 mm in­ternal diameter and 5 mm particle size. To extend the lifespan of this column we in­serted a safety column (RP8, Newguard, internal diameter 36x4.5 mm, particle size 7 mm, Brownlee) between the sample feeder unit and the analytical column, and we built in a cleansing column (CI8, internal diameter 36x4.5 mm, 20 mm particle size Rsil) between the pump and the sample feeder. To separate enantio­mers we utilised a two component gradient system with the following components: B: acetonitril in an A:40% methanol phosphate puffer (9.5 m M , pH:7.05). The speed of the flow was 1 cmVminute and gradient changed as a function of time like this:

Time (minute) 0 10 35 55 56 74 75

A % 95 95 83 72 67 67 U2

B % 5 5 17 2S 33 33 3 S

Detection - Maximum stimulus was 342 nm and maximal emission was 410 nm in the fluorescent detection of OPA/TATG derivatives.

Storage and evaluation of data - These were carried out utilising a LaChrom D-7000 System Manager device that made the presence of D-amino acids beside L ones measurable and évaluable even in extremely small concentration.

We evaluated the data produced by linear regression. D / L ratios and l n ( l + D / L ) / ( l - D / L ) of aspartic glutamic acids were plotted as a function of time by drawing so called calibrating diagrams.

RESULTS OF AGE E S T I M A T I O N BASED ON THE D-ASPARTIC , A N D D - G L U T A M I C A C I D CONTENTS OF TEETH

Our research project was carried out in collaboration with the Institute of Odontology and Earth Sciences Centre of Göteborg University. We analysed two recent tooth sample series to establish the so called calibrating diagrams. In 1998-1999 we gathered 22 teeth from the dental surgery of Pannon Agricultural University's Faculty of Animal Sciences in Kaposvár and we measured the D- and L-aspartic acid contents of them. When planning the sample we attempted to in­clude individuals with in the largest possible age envelope (17-62 years), and we also tried to select enough individuals from each age group to have a comprehen­sively representative sample.

Table 4 contains the data produced by analysing the Kaposvár dental sample of 1998. We calculated l n ( l + D / L ) ( l - D / L ) correlations both for aspartic and

Table 3. Individual sex and age data

Inventory No.

Grave No.

Sex Age esti­mated (year)

Inventory No. Grave No.

Sex Age esti­mated (year)

94.1.1. 1 male 35-39 94.1 .87. 82 male 40-44

94.1.3. 3 male 30-34 94.1 .92. 87 female 35-39

94.1.4. 4 A male 30-34 94.1 .94. 89 female 30-34

94.1.6. 5 male 45-49 94.1 .99. 93 male 30-34

94.1.7. 7 male 30-34 94.1 107 102 male 65-69

94.1.8. SA female 20-24 94.1 108 103 male 70-74

94.1.1 1. 9 male 25-29 94.1 110 104B male 45^19

94.1.13. 12 male 40-44 94.1 11 1 105 male 50-54

94.1.14. 13A female 45-49 94.1 112 106 female 65-69

94.1.17. 15A female 50-54 94.1 113 107 male 35-39

94.1.21. 18 female 35-39 94.1 ,115 109 male 35-39

94.1.22. 19A male ? 45-49 94.1 4 16 110 male 50-54

94.1.24. 20 male 45-49 94.1 117 1 1 1 female 55-59

94.1.26. 22 male 30-34 94.1 ,118 112 female 35-39

94.1.27. 23 female 35-39 94.1 .119 113 male 35-39

94.1.29., 30 25 (26) male 30-34 94.1 .122 117 female 35-39

94.1.32. 28 female 35-39 94.1 .123 118 female 35-39

94.1.40. 36A female 35-39 94.1 .127 122 female 30-39

94.1.43. 38 female 40-44 94.1 .129 124 male 45-^9

94.1.44. 39 male 35-39 94.1 .130 125 female 30-34

94.1.46. 4! male 50-54 94.1 .132 127 female 30-34

94.1.47. 42 male 40-44 94.1 .133 128 male 40^14

94.1.48. 43A male 40-44 94.1 .138 133 female 30-34

94.1.51. 45 female 45-49 94.1 .141 136 male 40^19

94.1.55. 49 male 35-39 94.1 .142 137 female ? 30-34

94.1.57. 51 male 40-44 94.1 .143 138 male 30-34

94.1.58. 52 male ? 35-39 94.1 .144 139 male '? 25-29

94.1.60. 54 male 25-34 94.1 .146 141 female 25-29

94.1.63. 57 female ? 25-29 94.1 .148 143 female ? 60-64

94.1.65. 59 female 35-39 94.1 .150 145 male 30-34

94.1.77. 72 male 65-69 94.1 .152 148 female 35-39

94.1.78. 73 male 35-39 94.1 .155 151 male ? 55-59

94.1.86. 81 male 30-34

glutamic acids besides D/L aspartic and D/L glutamic acid ratios. D / L ratios as well as the l n ( l + D / L ) ( l - D / L ) function were presented as a function of age. We cal­culated correlations of known ages and the D /L ratios of the two amino acids by linear regression. We found a very close positive relation between D/L ratio and age in case of aspartic acid contents. The value of r was 0.91 for the D /L ratio as well as for the calculated function. When analysing glutamic acid we concluded that the values of r fell between 0.98-0.99 for the D / L ratio as well for the calculated func­tion. Our examination of this dental sample of 22 teeth also led us to the conclusion that D-aspartic acid is a useful indicator for the estimation of individual age i f treated to the analytical methods (protein hydrolysis, derivative production, sepa­ration and identification of D- and L-enantiomers) we applied. We also drew the conclusion that D-glutamic acid content is also suitable for accurate age estimation beside D-aspartic acid, though D-glutamic acid is present in teeth in a smaller con­centration because of its different racemisation half-period. That is the reason why it is more difficult to measure and its scoring is a more demanding job for researchers.

Our first conclusions were based on a numerically small sample but we sup­ported them by analysing 102 dental samples in 1999. At the same time we opened up our field of research from comparatively young age groups towards older ones. Our 1999 examinations produced r:0.93 positive correlations between D / L ratios and individual ages both in case of D-aspartic and D-glutamic acids. The relation of an individual's age and the D-aspartic acid content of his or her tooth was pre­sented in Fig. 1, the correlation of age and D-glutamic acid was presented in Fig. 2. These two correlations are eminently suitable to estimate the age of any individual in the age envelope of 40-86 years on the basis of the D-aspartic and D-glutamic content of his or her tooth. The results of our 1999 research work confirmed those arrived at in 1998, so we may state the existence of an extremely close link be­tween any individual's age and the D-aspartic acid content of his or her tooth, and the D / L aspartic acid ratio on the basis of the analysis of a numerically large sample of teeth.

Another one of our assumptions was also proven correct: it was not just D-aspartic acid content but also D-glutamic acid content that could be used to esti­mate the age of an individual i f a sufficiently sensitive method of analysis was pro­vided to measure the small concentration of D-glutamic acid present.

In the third phase of our work we tried to apply the calibration diagrams pro­duced by our amino acid racemisation method on tooth samples originating in his­torical times. The age of individuals from the Kereki-Homokbánya Avar cemetery were estimated by the above mentioned anthropological methods in advance. The average of their age estimated by "traditional" anthropological methods, D /L as­partic and D/L glutamic ratios as well the age of these bone samples calculated on

the basis of D/L aspartic acid and D/L glutamic acid was presented in Table 5. In Fig. 3 we presented these ages by linear regression and this way it also presented the correlation of ages estimated by anthropological and by the two D-amino acid contents. It was evident from this figure that this correlation of ages estimated by the traditional and the new D-aspartic acid content methods was extremely close. When comparing the results of anthropological age estimation to those of the method based on amino acid racemisation the value of r exceeded 0.9 with both

Table 4. D amino acid content of teeth o f different age

Age (year) D / L ratio l n ( l + D / L ) / ( l - D / L )

Asp Glu Asp Glu

17 0.034 0.017 0.068 0.034

20 0.035 0.017 0.070 0.034

21 0.036 0.019 0.072 0.038

22 0.037 0.019 0.074 0.038

22 0.038 0.020 0.076 0.040

24 0.039 0.021 0.078 0.042

24 0.038 0.019 0.076 0.038

25 0.041 0.021 0.0821 0.042

27 0.042 0.021 0.0841 0.042

28 0.043 0.021 0.0861 0.042

31 0.044 0.022 0.0881 0.044

32 0.044 0.022 0.0881 0.044

35 0.047 0.024 0.0941 0.048

40 0.050 0.026 0.1001 0.052

42 0.052 0.026 0.1041 0.052

43 0.053 0.027 0.1061 0.054

43 0.053 0.028 0.1061 0.056

44 0.053 0.027 0.1061 0.054

46 0.055 0.028 0.1100 0.056

53 0.059 0.03 1 0.1 180 0.062

53 0.060 0.030 0.1201 0.060

62 0.065 0.033 0.1302 0.066

amino acids.

Annls hist.-nat. Mus. nam. tiling. 93, 2001

CONCLUSION

A method for the estimation of individual age on the basis of the quantity of D-aspartic and D-glutamic acid present in any tooth was developed. A calibration diagram, based on the analysis of 126 teeth from persons of 17-86 years of age, was drawn up. The D / L ratios and the l n ( l + D / L ) ( l - D / L ) correlation as a function of age was presented. We applied these calibration diagrams for the analysis of a single tooth from each and every one of 65 adult persons with unknown ages from

F i g . 1. Linear regression between the age o f life of the individuals and the D / L aspartic acid ratio o f their teeth

an Avar period cemetery. An extremely close link was found between ages esti­mated by anthropological methods and those produced by our method. We de­clared this close correlation of chemical - amino acid racemisation based - and an­thropologically estimated ages. We arrived at the conclusion that D- and L-aspartic acid analysed by our method was a suitable tool for the estimation of the age of any individual. What was more D- and L-glutamic acid was also proven to be suitable for the same purpose i f the necessary level of sensitivity was available in the ana­lytical technique utilised. The reliability of the estimation method could be im-

Fig . 2. Linear regression between the age of life of the individuals and the D / L glutamic acid ratio of their teeth

proved by using D-glutamic acid content too. On the other hand a significant dif­ference of the results produced by these two methods might indicate an error com­mitted by the researcher.

The significance of the anthropological utilisation of the amino acid racemisation based age estimation method was summarised under the following headings:

A single tooth or even a fragment with some enamel was sufficient for amino acid based analysis. It could be an especially useful characteristic when treating poorly preserved remains.

Fig . 3. Linear regression between the age o f life estimated by anthropological methods and D-amino acid content

Table 5. Comparison of the age estimated by anthropological methods and based on D-aspartic acid and D-glutamic acid content of the teeth

Inventory No.

Age estimated by anthropological methods (year)

D / L Asp Age estimated by D-aspartic acid content (year)

D / L Glu Age estimated by D-glutamic acid

content (year)

Males

94.1.1. 35-39 0.0429 36.8 0.0193 38.6

94.1.3. 30-34 0.0379 32.5 0.0163 32.6

94.1.4. 30-34 0.0365 31.3 0.0157 31.4

94.1.6. 45-49 0.0541 46.4 0.0249 49.8

94.1.7. 30-34 0.0354 30.3 0.0152 30.4

94.1.11. 25-29 0.0349 29.9 0.0142 28.4

94.1.13. 40-44 0.0499 42.7 0.0201 40.2

94.1.22. 45-49 0.0573 49.1 0.0252 50.4

94.1.24. 45-49 0.0546 46.8 0.0229 45.8

94.1.26. 30-34 0.0352 30.2 0.0149 29.8

94.1.29. 30-34 0.0383 32.8 0.0172 34.4

94.1.44. 35-39 0.0419 35.9 0.0199 39.8

94.1.46. 50-54 0.0636 54.5 0.0262 52.4

94.1.47. 40-44 0.0512 43.9 0.0199 39.8

94.1.48. 40^14 0.0493 42.2 0.0224 44.8

94.1.55. 35-39 0.0427 36.6 0.0193 38.6

94.1.57. 40-44 0.0499 42.8 0.0217 43.4

94.1.58. 35-39 0.0432 37.0 0.0191 38.2

94.1.60. 25-34 0.0340 29.1 0.0144 28.8

94.1.77. 65-69 0.0799 68.5 0.0346 69.2

94.1.78. 35-39 0.0421 36.1 0.0183 36.6

94.1.86. 30-34 0.0367 31.5 0.0164 32.8

94.1.87. 40-44 0.0522 44.7 0.0193 38.6

94.1.99. 30-34 0.0399 34.2 0.0151 30.2

94.1.107. 65-69 0.0834 71.5 0.0323 64.6

94.1.108. 70-74 0.0863 73.9 0.0366 73.2

94.1.110. 45-49 0.0532 45.6 0.0227 45.4

94.1.111. 50-54 0.0613 52.5 0.0242 48.4

Table 5 (continued)

Inventory Age estimated by D / L Asp Age estimated by D / L Glu Age estimated by No. anthropological D-aspartic acid D-glutamic acid

methods (year) content (year) content (year)

94.1.113. 35-39 0.0417 35.7 0.0182 36.4

94.1.115. 35-39 0.0442 37.8 0.0213 42.6

94.1.116. 50-54 0.0621 53.2 0.0247 49.4

94.1.119. 35-39 0.0474 40.6 0.0209 41.8

94.1.129. 45^19 0.0535 45.8 0.0234 46.8

94.1.133. 40-44 0.048 1 41.2 0.0209 41.8

94.1.141. 40-49 0.0547 46.9 0.0221 44.2

94.1.143. 30-34 0.0357 30.6 0.0156 31.2

94.1.144. 25-29 0.0329 28.2 0.0136 27.2

94.1.150. 30-34 0.0397 34.0 0.0171 34.2

94.1.155. 55-59 0.0692 59.3 0.0313 62.6

Females

94.1.8. 20-24 0.0256 21.9 0.0117 23.4

94.1.14. 45-49 0.0565 48.4 0.0234 46.8

94.1.17. 50-54 0.0613 52.5 0.0246 49.2

94.1.21. 35-39 0.0413 35.4 0.0197 39.4

94.1.27. 35-39 0.0446 38.2 0.0209 41.8

94.1.32. 35-39 0.0444 38.0 0.0193 38.6

94.1.43. 40^14 0.0519 44.4 0.0210 42.0

94.1.51. 45-49 0.0557 47.7 0.0224 44.8

94.1.63. 25-29 0.0312 26.7 0.0154 30.8

94.1.65. 35-39 0.0432 37.0 0.0229 45.8

94.1.92. 35-39 0.0413 35.4 0.0189 37.8

94.1.94. 30-34 0.0357 30.6 0.0156 31.2

94.1.112. 65-69 0.0798 67.6 0.0374 74.8

94.1.1 17. 55-59 0.0697 59.7 0.0269 53.8

94.1.1 18. 35-39 0.0473 40.5 0.0210 42.0

94.1.122. 35-39 0.0444 38.0 0.0219 43.8

94.1.123. 35-39 0.0417 35.7 0.0179 35.8

94.1.127. 30-39 0.0421 36.1 0.0186 37.2

Tab le 5 (continued)

Inventory Age estimated by D / L Asp Age estimated by D / L Glu Age estimated by No. anthropological D-aspartic acid D-glutamic acid

methods (year) content (year) content (year)

94.1 .130. 30-34 0.0381 32.6 0.0163 32.6

94.1 .132. 30-34 0.0373 32.0 0.0148 29.6

94.1 .138. 30-34 0.0357 30.6 0.0146 29.2

94.1 .140. 35-39 0.0421 36.1 0.0179 35.8

94.1 .142. 30-34 0.0387 33.2 0.0151 30.2

94.1 .146. 25-29 0.0302 25.9 0.0174 34.8

94.1 .148. 60-64 0.0742 63.6 0.0316 63.2

94.1 .152. 35-39 0.0473 40.5 0.0219 43.8

The amino acid racemisation based age estimation method was built on an ex­act foundation of natural sciences. Its application was clear-cut, and therefore free of intra- and interpersonal errors.

Amino acid racemisation provided help where it was the most urgently needed: in the estimation of age within the most difficult adult group.

The results produced by the amino acid racemisation based method presented a fine correlation to those of other "classic" anthropological methods. When used in combination they could confirm each-other's results.

For theoretical considerations this method was developed into a different en­tity compared to all routine age determination methods of anthropology. In con­trast to all the other methods, it did not take into consideration the genetically pro­grammed evolution of the organism, nor its responses to the environment or its physiological adaptation. It measured the structural alterations of amino acids, which are processes independent of circumstances of life and genetical facilities. In fact, amino acid racemisation measured the passing of chronological time and not that of biological time, and therefore it gave a completely new meaning to the word age in historical anthropology.

Acknowledgements - This paper was produced with the backing of Hungarian Scientific Re­search Fund ( O T K A grant Nos T-25023, F-026099 and F-02013).

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Catalogue of Palaearctic Diptera

edited by Á. SOÓS and L. PAPP

Volume 13 Anthomyiidae - Tachinidae

The Catalogue contains the basic taxonomic, nomenclatorial and distribution data of all species occurring in the Palaearctic Region wi th the fundamental morphological features for the ma­jority of the fly groups.

Volume 13 lists the names of 460 genera, 38 subgenera and 2389 species assigned to three families. Furthermore, 672 synony­mous generic and 2477 specific names, 1807 emendations, errors, nomina dubia and doubtful genera and species are listed. The pe­riod of the Catalogue extends from 1758 to 31 December, 1982.

Contents: Explication to distribution. Type-species designa­tions in Volume 13. New names proposed in Volume 13. Fam­ilies: Anthomyiidae (Á. D E L Y - D R A S K O V I T S ) . Rhinophoridae (B. H E R T I N G ) . Tachinidae (B. H E R T I N G and Á. D E L Y - D R A S K O V I T S ) .

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