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Estimation of age using aspartic acid racemisation in human dentin in Indian population
Forensic Science International 228 (2013) 38–41
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Estimation of age using aspartic acid racemisation in human dentin in Indian population S. Rajkumari a,*, Madhavan Nirmal b, P.M. Sunil b, A. Anton Smith c a
Sathyabama University Dental College and Hospital, Chennai, India Rajah Muthiah Dental College and Hospital, Annamalai University, Chidambaram, India c Department of Pharmacy, Annamalai University, Chidambaram, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 December 2012 Received in revised form 18 January 2013 Accepted 11 February 2013 Available online 16 March 2013
Aim: The quest to identify an accurate method of age estimation, had lead to the evaluation of aspartic acid racemisation in hard tissues of the human remains using high performance liquid chromatography (HPLC). Our study is aimed at the applicability of the racemisation technique to use dentin as the sample to estimate the age in South Indian sub-population. Materials and methods: Thirty-six non-carious teeth from living individuals distributed among 6 age groups (6 each), sexes (18 each) and jaws (18 each) were analysed for dextro (D) and levo (L) forms of aspartic acid using high performance liquid chromatography (HPLC) technique and their racemisation ratio were calculated for each tooth sample. Results: High correlation was obtained between the aspartic acid racemisation rates in dentin and age of the individual with an error limited to 3 years. Racemisation rates in teeth did not significantly differ between the sexes or jaws. Conclusion: The D-aspartic acid accumulation in dentin is synchronous with the aging of an individual and can be used as an accurate method of age estimation in our population. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Age estimation Teeth Dentin Racemisation Aspartic acid Forensic odontology
1. Introduction Ageing of the human organs involves intracellular and extracellular proteins that are subjected to a variety of nonenzymatic, spontaneous degradation reactions under physiologic conditions. The amino acids of vital tissue are L-amino acids and, with ageing, L-amino acids get converted to D-amino acids and accumulate in organs which show slow metabolism and this form of conversion is known as ‘‘racemisation’’ [1]. In principle, any amino acid residue contained in proteins might be analysed for enantiomeric purity, thus yielding racemisation data and hence valuable chronological information. The predominance of data for aspartic acid undoubtedly stems from its ease of isolation from protein hydrolysates and its relatively fast racemisation, approximately 0.055% inversion per year at 37 8C [2,3]. These factors combine to make aspartic acid a convenient choice for estimating the age of individuals [4]. The permanent proteins that are synthesised early in life are not subsequently exchanged and thus the D-aspartic acid gets accumulated. An age-dependent increase of D-aspartic acid has been
demonstrated in various tissues such as inter-vertebral discs, lens, brain, lung, bone and tooth [5]. Dentin has been the tissue of choice, as it forms early in the life of the individual, undergoes little biochemical turnover during life and, in total dentin and in the noncollagenous protein fraction of dentin, a very close relationship exists between age and the extent of aspartic acid racemisation [6]. The composition of the organic matrix of dentin is homogeneous enough to allow a reproducible age at death determination by analyzing total dentin or crude acidic dentinal extracts without further purification of the permanent proteins that show agedependent accumulation of D-aspartic acid. These optimum conditions are attributable to the generally very low protein turnover in dentin and this serves as the basis of a highly accurate method for age at death determination that has been applied successfully in forensic odontology [7]. The aim of the present study was to analyse the D/L ratio of aspartic acid in the dentin portion of the tooth thereby validate the usefulness of aspartic acid racemisation of dentin in age estimation among the South Indian sub-population by using high performance liquid chromatography (HPLC) technique. 2. Materials and methods
* Corresponding author. Tel.: +91 8122619788. E-mail addresses: [email protected], [email protected] (S. Rajkumari). 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.02.021
The sample consists of non-carious maxillary and mandibular, right and left first premolar teeth (n = 36) extracted for periodontal and orthodontic reasons, a tooth each from living individuals of age ranging from 11 years to 70 years, collected over
S. Rajkumari et al. / Forensic Science International 228 (2013) 38–41 a period of 7 months. The individual tooth sample was preserved in a dry state with a labelled cover and were not stored in any medium [8]. The samples were equally distributed among 2nd to 7th decade as 6 groups representing each decade (6 teeth each), between male and female (18 teeth each) and also between maxilla and mandible (18 teeth each). The samples were debrided and bucco-lingual longitudinal sections (1 mm) of dentin were prepared as described by Yekkala et al. [9] The dentinal samples were pulverised to fine powder using stainless steel mortar and pestle and demineralised with Na2 EDTA (0.5 M, pH 7.4) by shaking intensively for 2 h in a sonicator. Then it was centrifuged (5000 rpm for 5 min) to facilitate the separation of protein component. The sediment obtained was subjected to hydrolysis by hydrochloric acid (6 M) in a flame sealed tube at 100 8C in a water bath for 6 h. After hydrolysis, the samples were subjected to lyophilisation at 40 8C under vacuum of 200 mTorr for 6 h. The powdered dentinal samples were allowed to react with derivatizing solution of OPA-NAC (Sigma–Aldrich, St. Louis, USA), which was later subjected to high performance liquid chromatographic analysis. Standardisation of the procedure was done with reference materials for L-/Daspartic acid (Sigma–Aldrich, St. Louis, USA) before using the study samples. When the samples were evaluated in HPLC, the retention time for D-aspartic acid, Laspartic acid was 12 min and 14 min, respectively while OPA had a retention time of 8 min. A solvent peak was obtained in between the D- and L-aspartic acid peaks at 13 min, all the other compounds eluted after 15 min. The elution of different components in the sample solution was sensed by the fluorescence detector and analysed by TCA software (Totalchrom software 2.3, PerkinElmer, USA), which was read as a chromatograph. Significance of the assorted peaks at different intervals was compared with the standard chromatograph. For each sample, the area under the peak in the chromatograph (Fig. 1) was considered for both D- and L-aspartic acids from which D/L ratio was calculated. From these data, using least square method, the equation of the regression line defining the relationship between extent of racemisation and age (known) for each tooth was plotted. The age (unknown) of the sample was calculated from the obtained data by using the formula Ln[(1 + D/L)/(1 D/L)].
3. Results The actual age of the patient for each tooth sample, the D/L ratio, the Ln[(1 + D/L)/(1 D/L)] values and the estimated age are given in Table 1. Using Least square method, a plot of D/L ratio against the actual age to form the linear regression line also showed a significant relationship between the extent of racemisation and actual age with respect to each tooth (r = 0.9541) (Fig. 2a). Age is estimated from the linear regression line for age and Ln[(1 + D/L)/(1 D/L)](Fig. 2b). When the age is compared with the Ln[(1 + D/L)/(1 D/L)] a correlation coefficient value of 0.9543 was obtained which is statistically significant (Fig. 2b). When the percentage of error between the estimated age and the actual age was calculated, there existed an error of 7 years out of which 88.8% of results showed an error of 4 years (Fig. 3). The racemisation rates were analysed with least square method and plotted in a linear regression line for male and female, a
Table 1 The D/L ratio, the Ln[(1 + D/L)/(1 actual age for each sample. S.No.
Actual age
D/ L
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
11 12 14 15 18 20 22 22 24 27 28 30 32 34 36 38 39 40 41 43 44 45 48 50 52 54 55 56 57 58 62 63 65 68 70 70
0.0202 0.02 0.0209 0.022 0.0213 0.0226 0.023 0.0237 0.0241 0.025 0.0242 0.027 0.0301 0.0322 0.0338 0.0357 0.0349 0.0377 0.0368 0.0378 0.0388 0.0382 0.0392 0.0394 0.039 0.0399 0.0386 0.0408 0.0405 0.041 0.046 0.0428 0.0425 0.0462 0.0484 0.0516
39
D/L)]
ratio
and the estimated age are listed besides the
Ln[(1 + D/L)/ (1 D/L)]
Estimated age
Difference
0.0404 0.04 0.0418 0.044 0.0426 0.0452 0.0459 0.0474 0.0483 0.05 0.0484 0.054 0.0602 0.0646 0.0677 0.0715 0.0698 0.0755 0.0736 0.0758 0.0767 0.0764 0.0784 0.0789 00.078 0.0799 0.0772 0.0816 0.081 0.082 0.0857 0.085 0.092 0.0924 0.0968 0.1033
12 12 14 16 14 18 19 20 21 23 20 27 33 38 40 42 41 47 45 45 48 48 50 51 51 53 50 52 53 55 59 58 63 67 70 74
+1 0 0 +1 4 2 3 2 +2 4 6 3 +1 +4 +4 +4 +2 +7 +4 +2 +4 +3 +2 +1 1 1 5 2 4 3 +3 +5 2 +1 0 +4
correlation value of (r = 0.926) and (r = 0.962) were obtained respectively, with a male to female difference of 0.036 which was not statistically significant. Similarly a difference of 0.026 was obtained when racemisation rates were compared between teeth of maxilla and mandible. When the racemisation rates were compared between sexes (p = 0.485) and between jaws (p = 0.580), the results of t-tests did not show significant difference (pvalue > 0.01).
Fig. 1. A standard HPLC chromatograph of derivatised amino acids (L aspartic acid and
D
aspartic acid).
40
S. Rajkumari et al. / Forensic Science International 228 (2013) 38–41
Fig. 2. (a) A plot of D/L ratio of aspartic acid against the ages of teeth in the linear regression line. (b) A plot of Ln[(1 + D/L)/(1 the linear regression line.
D/L)]
Fig. 3. The percentage of errors between the estimated age and the actual age.
of aspartic acid against the ages of teeth in
S. Rajkumari et al. / Forensic Science International 228 (2013) 38–41
4. Discussion Estimation of age-at-death of both forensic and archaeological remains is one of the most significant problems in forensic odontology. The majority of existing morphological and histological techniques are highly inaccurate and show a bias towards underestimating the age of older individuals. One technique which has been successful in forensic age estimation is amino acid racemisation of hard tissue remains [6]. Most of these methods use gas chromatography where as, high performance liquid chromatography (HPLC) had revealed remarkable results in recent times [9,10]. Teeth were considered as optimal for the purpose of racemisation studies [11]. However, the different types of teeth show variation in their racemisation rate and thus the estimated age, which confirms that, age estimation based on the extent of racemisation in the same type of tooth yields better result than estimations from different types of tooth [12]. As dentin forms from crown towards the root apex, the D/L ratio should be higher in the crown and decrease towards the root apex. Therefore, Ohtani advocated the use of whole longitudinal sections rather than transverse ones, taken from the centre of the tooth because the racemisation varies from labial to lingual sides [12]. Among the different types of teeth, we have opted longitudinal dentinal sections of premolars since they are single rooted, small in size, and the maximum dentin is easily attainable. More so, the premolars are easier to collect as they are often sacrificed for orthodontic purposes. We observed a complete separation of D- and L-aspartic acid which was highly reproducible. The D/L ratio for younger age group was less, while that of the older age group was higher, suggesting that D/L aspartic acid ratio increases with age, which also signifies that the D-aspartic acid gets accumulated with age. The D/L values of our results were similar to that of Fu et al. [10], but differed from the study of Yekkala et al. [9] and Helfman and Bada [13]. The difference in the D/L ratio observed may be due to a difference in the specimens of dentin as well as the difference in analytical conditions used in the chromatographic and other analytical techniques [9]. It could also be considered that there is a possible influence of diverse factors such as the race, geographic location, dietary habits and other ethnic variables, since the study by Fu et al. which was similar to this study, was from Asia while the other studies were from Europe and USA [9,13]. The correlation coefficient r = 0.954 between the age and the ratio of D/L – enantiomers of aspartic acid in human dentin in our study, was almost similar to that observed by Ohtani et al. [12] in longitudinal sections of dentin (r = 0.961), Ohtani et al. [14] in cementum (r = 0.993–0.996,) Griffin et al. [6] in enamel (r = 0.92), Helfman and Bada [15] in enamel (r = 0.921) and in Ritz et al. [16] in total root dentin (r = 0.99). When the estimated age and the actual were compared there was an error of 0 to 7 years, of which 6 and 7 years had occurred once, and the mean of the errors show an error limited to 3 years, similar to Yekkala et al. [9] and Ohtani [8,12]. The possible explanation for the error could be the technical limitation on the purity of the dentin sample [10].
41
The racemisation rates of dentin did not show any significant variation between the sexes, which contradicts the study on racemisation rates in alveolar bone, where the females showed less racemisation rate than males [17]. The variation in D/L ratio in alveolar bone is due to increased metabolic turnover; while teeth being slow metabolising tissue, they may not show any differences between the sexes. Since the teeth collected were from both maxilla and mandible, an attempt was made to rule out the possible difference in racemisation rates between the jaws. No such differences in correlation coefficient existed as confirmed by least square method and t-test (p = 0.580). To conclude, the results of our study indicate that the D-aspartic acid accumulation in dentin is synchronous with the aging of an individual and emphasises that aspartic acid racemisation estimated in dentin can be used as an accurate method of age estimation in our population. References [1] S. Ohtani, Estimation of age from dentin by utilizing the racemization of aspartic acid: influence of pH, Forensic Sci. Int. 75 (1995) 181–187. [2] T. Ogino, H. Ogino, B. Nagy, Application of aspartic acid racemization to forensic odontology: post mortem designation of age at death, Forensic Sci. Int. 29 (1985) 259–267. [3] Shimoyama, K. Harada, An age determination of an ancient burial mound man by apparent racemization reaction of aspartic acid in tooth dentine, Chem. Lett. 10 (1984) 1661–1664. [4] R.D. Gillard, A.M. Pollard, P.A. Sutton, D.K. Whittaker, An improved method for age at death determination from the measurement of D-aspartic acid in dental collagen, Archaeometry 32 (1990) 61–70. [5] C.R. McCudden, V.B. Kraus, Biochemistry of amino acid racemization and clinical application to musculoskeletal disease, Clin. Biochem. 39 (December (12)) (2006) 1112–1130. [6] R.C. Griffin, H. Moody, K.E. Penkman, M.J. Collins, The application of amino acid racemization in the acid soluble fraction of enamel to the estimation of the age of human teeth, Forensic Sci. Int. 175 (2008) 11–16. [7] S. Ritz, A. Turzynski, H.W. Schu¨tz, A. Hollmann, G. Rochholz, Identification of osteocalcin as a permanent aging constituent of the bone matrix: basis for an accurate age at death determination, Forensic Sci. Int. 77 (January (1–2)) (1996) 13–26. [8] S. Ohtani, Estimation of age from dentin by using the racemisation reaction of aspartic acid, Am. J. Forensic Med. Pathol. 16 (1995) 158–161. [9] R. Yekkala, C. Meers, A. Van Schepdael, J. Hoogmartens, I. Lambrichts, G. Willems, Racemization of aspartic acid from human dentin in the estimation of chronological age, Forensic Sci. Int. 159 (May (Suppl. 1)) (2006) S89–S94. [10] S.J. Fu, C.C. Fan, H.W. Song, F.Q. Wei, Age estimation using a modified HPLC determination of ratio of aspartic acid in dentin, Forensic Sci. Int. 73 (1995) 35–40. [11] J. Sajdok, A. Pilin, F. Pudil, J. Zı´dkova´, J. Ka´s, Preliminary communication. A new method of age estimation based on the changes in human non-collagenous proteins from dentin, Forensic Sci. Int. 156 (2006) 245–249. [12] S. Ohtani, R. Ito, T. Yamamoto, Differences in the D/L aspartic acid ratios in dentin among different types of teeth from the same individual and estimated age, Int. J. Legal Med. 117 (June (3)) (2003) 149–152. [13] P.M. Helfman, J.L. Bada, Aspartic acid racemization in tooth enamel from living humans, Proc. Natl. Acad. Sci. U.S.A. 72 (1975) 2891–2894. [14] S. Ohtani, H. Sugimoto, H. Sugeno, S. Yamamoto, K. Yamamoto, Racemization of aspartic acid in human cementum with age, Arch. Oral Biol. 40 (February (2)) (1995) 91–95. [15] P.M. Helfman, J.L. Bada, Aspartic acid racemization in dentine as a measure of ageing, Nature 262 (1976) 279–281. [16] S. Ritz, H.W. Schu¨tz, C. Peper, Postmortem estimation of age at death based on aspartic acid racemization in dentin: its applicability for root dentin, Int. J. Legal Med. 105 (5) (1993) 289–293. [17] S. Ohtani, T. Yamamoto, I. Abe, Y. Kinoshita, Age-dependent changes in the racemisation ratio of aspartic acid in human alveolar bone, Arch. Oral Biol. 52 (2007) 233–236.
Contents lists available at SciVerse ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Estimation of age using aspartic acid racemisation in human dentin in Indian population S. Rajkumari a,*, Madhavan Nirmal b, P.M. Sunil b, A. Anton Smith c a
Sathyabama University Dental College and Hospital, Chennai, India Rajah Muthiah Dental College and Hospital, Annamalai University, Chidambaram, India c Department of Pharmacy, Annamalai University, Chidambaram, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 December 2012 Received in revised form 18 January 2013 Accepted 11 February 2013 Available online 16 March 2013
Aim: The quest to identify an accurate method of age estimation, had lead to the evaluation of aspartic acid racemisation in hard tissues of the human remains using high performance liquid chromatography (HPLC). Our study is aimed at the applicability of the racemisation technique to use dentin as the sample to estimate the age in South Indian sub-population. Materials and methods: Thirty-six non-carious teeth from living individuals distributed among 6 age groups (6 each), sexes (18 each) and jaws (18 each) were analysed for dextro (D) and levo (L) forms of aspartic acid using high performance liquid chromatography (HPLC) technique and their racemisation ratio were calculated for each tooth sample. Results: High correlation was obtained between the aspartic acid racemisation rates in dentin and age of the individual with an error limited to 3 years. Racemisation rates in teeth did not significantly differ between the sexes or jaws. Conclusion: The D-aspartic acid accumulation in dentin is synchronous with the aging of an individual and can be used as an accurate method of age estimation in our population. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Age estimation Teeth Dentin Racemisation Aspartic acid Forensic odontology
1. Introduction Ageing of the human organs involves intracellular and extracellular proteins that are subjected to a variety of nonenzymatic, spontaneous degradation reactions under physiologic conditions. The amino acids of vital tissue are L-amino acids and, with ageing, L-amino acids get converted to D-amino acids and accumulate in organs which show slow metabolism and this form of conversion is known as ‘‘racemisation’’ [1]. In principle, any amino acid residue contained in proteins might be analysed for enantiomeric purity, thus yielding racemisation data and hence valuable chronological information. The predominance of data for aspartic acid undoubtedly stems from its ease of isolation from protein hydrolysates and its relatively fast racemisation, approximately 0.055% inversion per year at 37 8C [2,3]. These factors combine to make aspartic acid a convenient choice for estimating the age of individuals [4]. The permanent proteins that are synthesised early in life are not subsequently exchanged and thus the D-aspartic acid gets accumulated. An age-dependent increase of D-aspartic acid has been
demonstrated in various tissues such as inter-vertebral discs, lens, brain, lung, bone and tooth [5]. Dentin has been the tissue of choice, as it forms early in the life of the individual, undergoes little biochemical turnover during life and, in total dentin and in the noncollagenous protein fraction of dentin, a very close relationship exists between age and the extent of aspartic acid racemisation [6]. The composition of the organic matrix of dentin is homogeneous enough to allow a reproducible age at death determination by analyzing total dentin or crude acidic dentinal extracts without further purification of the permanent proteins that show agedependent accumulation of D-aspartic acid. These optimum conditions are attributable to the generally very low protein turnover in dentin and this serves as the basis of a highly accurate method for age at death determination that has been applied successfully in forensic odontology [7]. The aim of the present study was to analyse the D/L ratio of aspartic acid in the dentin portion of the tooth thereby validate the usefulness of aspartic acid racemisation of dentin in age estimation among the South Indian sub-population by using high performance liquid chromatography (HPLC) technique. 2. Materials and methods
* Corresponding author. Tel.: +91 8122619788. E-mail addresses: [email protected], [email protected] (S. Rajkumari). 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.02.021
The sample consists of non-carious maxillary and mandibular, right and left first premolar teeth (n = 36) extracted for periodontal and orthodontic reasons, a tooth each from living individuals of age ranging from 11 years to 70 years, collected over
S. Rajkumari et al. / Forensic Science International 228 (2013) 38–41 a period of 7 months. The individual tooth sample was preserved in a dry state with a labelled cover and were not stored in any medium [8]. The samples were equally distributed among 2nd to 7th decade as 6 groups representing each decade (6 teeth each), between male and female (18 teeth each) and also between maxilla and mandible (18 teeth each). The samples were debrided and bucco-lingual longitudinal sections (1 mm) of dentin were prepared as described by Yekkala et al. [9] The dentinal samples were pulverised to fine powder using stainless steel mortar and pestle and demineralised with Na2 EDTA (0.5 M, pH 7.4) by shaking intensively for 2 h in a sonicator. Then it was centrifuged (5000 rpm for 5 min) to facilitate the separation of protein component. The sediment obtained was subjected to hydrolysis by hydrochloric acid (6 M) in a flame sealed tube at 100 8C in a water bath for 6 h. After hydrolysis, the samples were subjected to lyophilisation at 40 8C under vacuum of 200 mTorr for 6 h. The powdered dentinal samples were allowed to react with derivatizing solution of OPA-NAC (Sigma–Aldrich, St. Louis, USA), which was later subjected to high performance liquid chromatographic analysis. Standardisation of the procedure was done with reference materials for L-/Daspartic acid (Sigma–Aldrich, St. Louis, USA) before using the study samples. When the samples were evaluated in HPLC, the retention time for D-aspartic acid, Laspartic acid was 12 min and 14 min, respectively while OPA had a retention time of 8 min. A solvent peak was obtained in between the D- and L-aspartic acid peaks at 13 min, all the other compounds eluted after 15 min. The elution of different components in the sample solution was sensed by the fluorescence detector and analysed by TCA software (Totalchrom software 2.3, PerkinElmer, USA), which was read as a chromatograph. Significance of the assorted peaks at different intervals was compared with the standard chromatograph. For each sample, the area under the peak in the chromatograph (Fig. 1) was considered for both D- and L-aspartic acids from which D/L ratio was calculated. From these data, using least square method, the equation of the regression line defining the relationship between extent of racemisation and age (known) for each tooth was plotted. The age (unknown) of the sample was calculated from the obtained data by using the formula Ln[(1 + D/L)/(1 D/L)].
3. Results The actual age of the patient for each tooth sample, the D/L ratio, the Ln[(1 + D/L)/(1 D/L)] values and the estimated age are given in Table 1. Using Least square method, a plot of D/L ratio against the actual age to form the linear regression line also showed a significant relationship between the extent of racemisation and actual age with respect to each tooth (r = 0.9541) (Fig. 2a). Age is estimated from the linear regression line for age and Ln[(1 + D/L)/(1 D/L)](Fig. 2b). When the age is compared with the Ln[(1 + D/L)/(1 D/L)] a correlation coefficient value of 0.9543 was obtained which is statistically significant (Fig. 2b). When the percentage of error between the estimated age and the actual age was calculated, there existed an error of 7 years out of which 88.8% of results showed an error of 4 years (Fig. 3). The racemisation rates were analysed with least square method and plotted in a linear regression line for male and female, a
Table 1 The D/L ratio, the Ln[(1 + D/L)/(1 actual age for each sample. S.No.
Actual age
D/ L
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
11 12 14 15 18 20 22 22 24 27 28 30 32 34 36 38 39 40 41 43 44 45 48 50 52 54 55 56 57 58 62 63 65 68 70 70
0.0202 0.02 0.0209 0.022 0.0213 0.0226 0.023 0.0237 0.0241 0.025 0.0242 0.027 0.0301 0.0322 0.0338 0.0357 0.0349 0.0377 0.0368 0.0378 0.0388 0.0382 0.0392 0.0394 0.039 0.0399 0.0386 0.0408 0.0405 0.041 0.046 0.0428 0.0425 0.0462 0.0484 0.0516
39
D/L)]
ratio
and the estimated age are listed besides the
Ln[(1 + D/L)/ (1 D/L)]
Estimated age
Difference
0.0404 0.04 0.0418 0.044 0.0426 0.0452 0.0459 0.0474 0.0483 0.05 0.0484 0.054 0.0602 0.0646 0.0677 0.0715 0.0698 0.0755 0.0736 0.0758 0.0767 0.0764 0.0784 0.0789 00.078 0.0799 0.0772 0.0816 0.081 0.082 0.0857 0.085 0.092 0.0924 0.0968 0.1033
12 12 14 16 14 18 19 20 21 23 20 27 33 38 40 42 41 47 45 45 48 48 50 51 51 53 50 52 53 55 59 58 63 67 70 74
+1 0 0 +1 4 2 3 2 +2 4 6 3 +1 +4 +4 +4 +2 +7 +4 +2 +4 +3 +2 +1 1 1 5 2 4 3 +3 +5 2 +1 0 +4
correlation value of (r = 0.926) and (r = 0.962) were obtained respectively, with a male to female difference of 0.036 which was not statistically significant. Similarly a difference of 0.026 was obtained when racemisation rates were compared between teeth of maxilla and mandible. When the racemisation rates were compared between sexes (p = 0.485) and between jaws (p = 0.580), the results of t-tests did not show significant difference (pvalue > 0.01).
Fig. 1. A standard HPLC chromatograph of derivatised amino acids (L aspartic acid and
D
aspartic acid).
40
S. Rajkumari et al. / Forensic Science International 228 (2013) 38–41
Fig. 2. (a) A plot of D/L ratio of aspartic acid against the ages of teeth in the linear regression line. (b) A plot of Ln[(1 + D/L)/(1 the linear regression line.
D/L)]
Fig. 3. The percentage of errors between the estimated age and the actual age.
of aspartic acid against the ages of teeth in
S. Rajkumari et al. / Forensic Science International 228 (2013) 38–41
4. Discussion Estimation of age-at-death of both forensic and archaeological remains is one of the most significant problems in forensic odontology. The majority of existing morphological and histological techniques are highly inaccurate and show a bias towards underestimating the age of older individuals. One technique which has been successful in forensic age estimation is amino acid racemisation of hard tissue remains [6]. Most of these methods use gas chromatography where as, high performance liquid chromatography (HPLC) had revealed remarkable results in recent times [9,10]. Teeth were considered as optimal for the purpose of racemisation studies [11]. However, the different types of teeth show variation in their racemisation rate and thus the estimated age, which confirms that, age estimation based on the extent of racemisation in the same type of tooth yields better result than estimations from different types of tooth [12]. As dentin forms from crown towards the root apex, the D/L ratio should be higher in the crown and decrease towards the root apex. Therefore, Ohtani advocated the use of whole longitudinal sections rather than transverse ones, taken from the centre of the tooth because the racemisation varies from labial to lingual sides [12]. Among the different types of teeth, we have opted longitudinal dentinal sections of premolars since they are single rooted, small in size, and the maximum dentin is easily attainable. More so, the premolars are easier to collect as they are often sacrificed for orthodontic purposes. We observed a complete separation of D- and L-aspartic acid which was highly reproducible. The D/L ratio for younger age group was less, while that of the older age group was higher, suggesting that D/L aspartic acid ratio increases with age, which also signifies that the D-aspartic acid gets accumulated with age. The D/L values of our results were similar to that of Fu et al. [10], but differed from the study of Yekkala et al. [9] and Helfman and Bada [13]. The difference in the D/L ratio observed may be due to a difference in the specimens of dentin as well as the difference in analytical conditions used in the chromatographic and other analytical techniques [9]. It could also be considered that there is a possible influence of diverse factors such as the race, geographic location, dietary habits and other ethnic variables, since the study by Fu et al. which was similar to this study, was from Asia while the other studies were from Europe and USA [9,13]. The correlation coefficient r = 0.954 between the age and the ratio of D/L – enantiomers of aspartic acid in human dentin in our study, was almost similar to that observed by Ohtani et al. [12] in longitudinal sections of dentin (r = 0.961), Ohtani et al. [14] in cementum (r = 0.993–0.996,) Griffin et al. [6] in enamel (r = 0.92), Helfman and Bada [15] in enamel (r = 0.921) and in Ritz et al. [16] in total root dentin (r = 0.99). When the estimated age and the actual were compared there was an error of 0 to 7 years, of which 6 and 7 years had occurred once, and the mean of the errors show an error limited to 3 years, similar to Yekkala et al. [9] and Ohtani [8,12]. The possible explanation for the error could be the technical limitation on the purity of the dentin sample [10].
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The racemisation rates of dentin did not show any significant variation between the sexes, which contradicts the study on racemisation rates in alveolar bone, where the females showed less racemisation rate than males [17]. The variation in D/L ratio in alveolar bone is due to increased metabolic turnover; while teeth being slow metabolising tissue, they may not show any differences between the sexes. Since the teeth collected were from both maxilla and mandible, an attempt was made to rule out the possible difference in racemisation rates between the jaws. No such differences in correlation coefficient existed as confirmed by least square method and t-test (p = 0.580). To conclude, the results of our study indicate that the D-aspartic acid accumulation in dentin is synchronous with the aging of an individual and emphasises that aspartic acid racemisation estimated in dentin can be used as an accurate method of age estimation in our population. References [1] S. Ohtani, Estimation of age from dentin by utilizing the racemization of aspartic acid: influence of pH, Forensic Sci. Int. 75 (1995) 181–187. [2] T. Ogino, H. Ogino, B. Nagy, Application of aspartic acid racemization to forensic odontology: post mortem designation of age at death, Forensic Sci. Int. 29 (1985) 259–267. [3] Shimoyama, K. Harada, An age determination of an ancient burial mound man by apparent racemization reaction of aspartic acid in tooth dentine, Chem. Lett. 10 (1984) 1661–1664. [4] R.D. Gillard, A.M. Pollard, P.A. Sutton, D.K. Whittaker, An improved method for age at death determination from the measurement of D-aspartic acid in dental collagen, Archaeometry 32 (1990) 61–70. [5] C.R. McCudden, V.B. Kraus, Biochemistry of amino acid racemization and clinical application to musculoskeletal disease, Clin. Biochem. 39 (December (12)) (2006) 1112–1130. [6] R.C. Griffin, H. Moody, K.E. Penkman, M.J. Collins, The application of amino acid racemization in the acid soluble fraction of enamel to the estimation of the age of human teeth, Forensic Sci. Int. 175 (2008) 11–16. [7] S. Ritz, A. Turzynski, H.W. Schu¨tz, A. Hollmann, G. Rochholz, Identification of osteocalcin as a permanent aging constituent of the bone matrix: basis for an accurate age at death determination, Forensic Sci. Int. 77 (January (1–2)) (1996) 13–26. [8] S. Ohtani, Estimation of age from dentin by using the racemisation reaction of aspartic acid, Am. J. Forensic Med. Pathol. 16 (1995) 158–161. [9] R. Yekkala, C. Meers, A. Van Schepdael, J. Hoogmartens, I. Lambrichts, G. Willems, Racemization of aspartic acid from human dentin in the estimation of chronological age, Forensic Sci. Int. 159 (May (Suppl. 1)) (2006) S89–S94. [10] S.J. Fu, C.C. Fan, H.W. Song, F.Q. Wei, Age estimation using a modified HPLC determination of ratio of aspartic acid in dentin, Forensic Sci. Int. 73 (1995) 35–40. [11] J. Sajdok, A. Pilin, F. Pudil, J. Zı´dkova´, J. Ka´s, Preliminary communication. A new method of age estimation based on the changes in human non-collagenous proteins from dentin, Forensic Sci. Int. 156 (2006) 245–249. [12] S. Ohtani, R. Ito, T. Yamamoto, Differences in the D/L aspartic acid ratios in dentin among different types of teeth from the same individual and estimated age, Int. J. Legal Med. 117 (June (3)) (2003) 149–152. [13] P.M. Helfman, J.L. Bada, Aspartic acid racemization in tooth enamel from living humans, Proc. Natl. Acad. Sci. U.S.A. 72 (1975) 2891–2894. [14] S. Ohtani, H. Sugimoto, H. Sugeno, S. Yamamoto, K. Yamamoto, Racemization of aspartic acid in human cementum with age, Arch. Oral Biol. 40 (February (2)) (1995) 91–95. [15] P.M. Helfman, J.L. Bada, Aspartic acid racemization in dentine as a measure of ageing, Nature 262 (1976) 279–281. [16] S. Ritz, H.W. Schu¨tz, C. Peper, Postmortem estimation of age at death based on aspartic acid racemization in dentin: its applicability for root dentin, Int. J. Legal Med. 105 (5) (1993) 289–293. [17] S. Ohtani, T. Yamamoto, I. Abe, Y. Kinoshita, Age-dependent changes in the racemisation ratio of aspartic acid in human alveolar bone, Arch. Oral Biol. 52 (2007) 233–236.