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Med Sci Law OnlineFirst, published on March 3, 2014 as doi:10.1177/0025802414524383
Original article
Racemization of aspartic acid in root dentin as a tool for age estimation in a Kuwaiti population
Medicine, Science and the Law 0(0) 1–8 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0025802414524383 msl.sagepub.com
Mohamed Amin Elfawal1, Sahib Issa Alqattan2 and Noha Ayman Ghallab3
Abstract Estimation of age is one of the most significant tasks in forensic practice. Amino acid racemization is considered one of the most reliable and accurate methods of age estimation and aspartic acid shows a high racemization reaction rate. The present study has investigated the application of aspartic acid racemization in age estimation in a Kuwaiti population using root dentin from a total of 89 upper first premolar teeth. The D/L ratio of aspartic acid was obtained by HPLC technique in a test group of 50 subjects and a linear regression line was established between aspartic acid racemization and age. The correlation coefficient (r) was 0.97, and the standard error of estimation was 1.26 years. The racemization age ‘‘t’’ of each subject was calculated by applying the following formula: ln [(1 þ D/L)/(1 D/L)] ¼ 0.003181 t þ (0.01591). When the proposed formula ‘‘estimated age t ¼ ln [(1 þ D/L)/(1 D/L)] þ 0.01591/0.003181’’ was applied to a validation group of 39 subjects, the range of error was less than one year in 82.1% of the cases and the standard error of estimation was 1.12. The current work has established a reasonably significant correlation of the D-/L-aspartic acid ratio with age, and proposed an apparently reliable formula for calculating the age in Kuwaiti populations through aspartic acid racemization. Further research is required to find out whether similar findings are applicable to other ethnic populations. Keywords age estimation, aspartic acid, racemization, teeth
Introduction Identification is one of the important functions in forensic work. Among establishing many aspects of identification of a human body, estimation of age has imperative significance. Age estimation in living subjects, cadavers, and human remains may elucidate matters with considerable legal and social consequences. The implications of a ‘‘lack of identification’’ of a cadaver cannot be underrated. The number of unidentified cadavers and human remains throughout the world is on the increase.1 Teeth, the hardest of all human tissues, are highly resistant to physical and chemical influences. Even in cases of extensive destruction of the body, the teeth are often preserved, especially the molars.2 Meanwhile, there are legal circumstances requiring age estimation in living individuals without valid proof of date of birth e.g. adolescent drug smugglers in Asian and Gulf countries. Determination of age using teeth have been studied by numerous workers and updated with new techniques, ranging from conventional morphological methods3,4 to biochemical analytical techniques.5
Age estimation in children and adolescents frequently depends on radiological examination of skeletal and dental development. In adults, however, such traditional methods for ageing are highly subjective.6–8 Conversely, aspartic acid racemization (AAR) is considered one of the most reliable and accurate methods of age estimation in forensic practice.8–13 Racemization is a chemical reaction in which spontaneous conversion of L-enantiomer into D-enantiomer occurs in the course of ageing and aspartic acid (Asp) shows a high racemization reaction rate.14 Throughout time, such L-amino acids are partly converted to D-enantiomers until an equilibrium mixture of both is attained.15 Consequently, measurement of 1
Faculty of Medicine, Kuwait University, Saffat, Kuwait Ministry of Interior Kuwait, Kuwait 3 Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt 2
Corresponding author: Mohamed Amin Elfawal, Faculty of Medicine, Kuwait University, Saffat, Kuwait, 13110 Kuwait. Email: [email protected]
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the D:L Asp ratio will give an indication of the time elapsed since the dentin was laid down. Racemization takes place in tissues with slow metabolism, such as teeth, bones, cartilages, eye-lens, or brain.16–19 Nevertheless, employing non-dental tissues, e.g. cartilage, bone, or elastin, in age estimation by AAR has shown a less significant correlation compared to dentinal proteins.20–22 The inter-conversion of Asp enantiomers is a reversible first-order chemical reaction. It is influenced by several factors such as type of tooth, analyzed, tooth part, surrounding temperature, humidity, pH, and fixative used.23,24 It has been found that the earlier the tooth formation is accomplished, the higher the level of D-Asp. Accordingly, different types of teeth of the same individual, formed throughout different periods of growth, would have varied levels of D-Asp.14 Hence, a specific type of tooth is required to be employed as a standard reference and is expected to yield better results than the use of various types of teeth for age estimation in medico-legal practice.25 In the present study, we have investigated the application of AAR in root dentin from upper first premolar teeth as a tool in age estimation in a Kuwaiti population.
Materials and methods Chemicals Powders of D- and L-aspartic acid, O-phthaldialdehyde (OPA), N-acetyl-L-cysteine (NAC), phenylmethyl sulfonyl fluoride, and alpha toluene sulfonel fluoride were all purchased from Sigma-Aldrich (USA).
Reagent preparation OPA / NAC reagent was prepared by dissolving OPA (8 mg) in methanol (600 mL), and then 500 mL of 0.4 M Na borate buffer (pH 9.4) and 120 mL of 1 M NAC were added. The reagent stock solution was stored at 4 C.
Instrumentation and operating procedures The HPLC system (Waters, Milford, Massachusetts, USA) consisted of 600S Controller, 626 Pump Pressure, 717 Plus Autosampler and 474 Scanning Fluorescence Detector. The fluorescence detection was used at ex 340 nm and em 420 nm. The pump pressure was maintained at 1800 psi. Research analytical oven was used for hydrolysis purposes (Oldham, Lancs, England). A Kromasil C8, 5 mm chiral column (4.6 250 mm) (USA) was used for separation of D- and L-Asp. A gradient elution was carried out with two solutions: A and B. Solution A was made of 50 mM sodium acetate pH 5.9 and solution B was made of 80% methanol and 20% of
solution A. During the first 5 minutes the elution was 90% of solution A and 10% of solution B, then the percentage of solution B was increased up to 100% linearly over a period of 5 min. The flow rate of the mobile phase was 1 mL/min and the injection volume was 10 mL.
Sample preparation and derivatization The method used in this work was similar to those employed by Yekkala et al.25 and Fu et al.26 A total of 89 upper first premolar teeth were collected. They were divided into two groups, a test group included 50 teeth (33 males and 17 females) of known age (age range between 10 and 31 years and mean age 14.98 4.76), and a validation group consisted of 39 teeth (26 males and 13 females) of recorded age, but unknown to the investigator (age range between 10 and 30 years and mean age 15.1 5.16). All teeth studied were sound, free from restorations and dental caries, and were extracted from living Kuwaiti individuals because of periodontal diseases or orthodontic reasons. Recent reports have shown that periodontal disease is the commonest underlying cause of teeth extraction in Kuwaiti adults.27 Teeth were stored frozen-dry to avoid the influence of fixatives.21,28 The crown and apical 1/3 of the root were separated using dental diamond disc and bur, both cooled in liquid nitrogen in order to avoid heatrelated racemization of Asp. The dentin samples were broken into small shards, crushed into powder, and 40–80 mg were washed in 15% NaCl at 4 C overnight, centrifuged (13000 r/min for 5 min) three times, and dried by a cold system model and a high vacuum pump. The dry residue was re-suspended in 1 mL of 6 M HCl and hydrolyzed for 6 hours. The hydrolyzates were dried by the same vacuum pump apparatus, desalted on a cation exchange column, and 1 mL of double distilled water was added. The dentin samples which contained the amino acids were passed down the column. Finally, 6 mL of 2 M ammonium hydroxide solution were used to elute the amino acids which were then collected in a clean glass tube. Four of 1.5 mL aliquots were made and then derivatized.29–31 One of the four aliquots was reconstituted with double distilled water for derivatization and the other three were stored for further analysis. Derivatization was accomplished by mixing 10 mL of the prepared sample solution with 20 mL of OPANAC derivatization reagent. After 2.5 minutes, 200 mL of 50 mmol Na acetate was added, and then 10 mL of the derivatized solution was injected into the HPLC system. The HPLC analysis was done under ambient temperature of 23 C. The total run time was 14 minutes and the retention time for D-Asp was 9.767 minutes and that for L-Asp was 11.683 minutes (Figure 1). A control was made using water, instead of the dentin sample, under the same conditions and gave no reaction.
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The analysis of the D/L ratio for Asp in the dentin samples was calculated. The formula ln [(1 þ D/L) / (1 D/L)] was employed.32 The estimated age was assessed from the D/L ratio based on the fact that racemization of Asp follows a first order reversible rate law equation: ln [(1 þ D/L) / (1 D/ L)] ¼ 2kt þ A,33 where D/L is the ratio of the area of eluted peaks of D and L Asp, 2k is the constant ‘‘slope’’ of racemization, which can be calculated from a linear regression of ln(1þD/L) / (1-D/L)] versus age of the individual, t is the estimated age (years), and A is the intercept of the linear regression line with the Y axis.
investigator. According to the proposed formula, the age was calculated as follows: Estimated age t ¼
ln ½ð1 þ D=LÞ=ð1 D=LÞ þ 0:01591 0:003181
Table 2 and Figure 3 illustrate the comparison between the actual and calculated age, employing the proposed formula, among the validation group. Table 3 shows the distribution of error values in the
0.1 Series1
Results
0.09
Linear (Series1)
0.08
ln [(1+D/L) / (1-D/L)]
The root dentin of 50 upper first premolars (test group) was analyzed and the D/L ratio of Asp was obtained. A linear regression line was established, which is a plot of ln [(1 þ D/L) / (1 D/L)] of Asp versus the known age (Figure 2). It was used to calculate the 2k and A values for the kinetic equation of racemization of amino acids: ln [(1 þ D/L) / (1 D/ L)] ¼ 2kt þ A. Accordingly, the racemization age ‘‘t’’ of each subject was calculated by applying the following formula: ln [(1 þ D/L)/(1 D/L)] ¼ 0.003181 t þ (0.01591). The correlation coefficient value (r) was 0.97 and the coefficient of determination (r2) was 0.92. The standard error of estimation, calculated by descriptive analysis, was 1.26 years (actual age minus racemization age). Table 1 shows the experimental data and racemization age for the test group. The proposed formula was applied to a validation group of 39 upper first premolars, where the age of the subject has been recorded but was unknown to the
0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0
10
20
30
40
Age (years)
Figure 2. Linear regression line for the test group (total number 50 premolars).
Figure 1. HPLC chromatogram of aspartic acid separation (C ¼ 1 mg/mL methanol).
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Table 1. Experimental data and racemization age estimation for the test group (50 upper first premolar teeth). Actual age
D/L
ln [(1 þ D/L)/ (1 D/L)]
Racemization age
1
16
0.0157
0.0315
15
1
2
14
0.0128
0.0256
13
1
3
15
0.0168
0.0336
16
1
4
12
0.0110
0.0221
12
0
5
15
0.0160
0.0320
15
0
6
14
0.0143
0.0287
14
0
7
11
0.0094
0.0188
11
0
8
17
0.0198
0.0396
17
0
9
15
0.0171
0.0343
16
1
No.
Error (years)
10
14
0.0153
0.0306
15
1
11
10
0.0050
0.0099
8
2
12
10
0.0061
0.0122
9
1
13
11
0.0091
0.0182
11
0
14
11
0.0095
0.0190
11
0
15
11
0.0098
0.0195
11
0
16
22
0.0323
0.0645
25
3
17
13
0.0120
0.0240
13
0
18
14
0.0152
0.0305
15
1
19
30
0.0350
0.0699
27
3
20
10
0.0084
0.0167
10
0
21
15
0.0172
0.0344
16
1
22
11
0.0107
0.0213
12
1
23
31
0.0444
0.0889
33
2
24
13
0.0126
0.0251
13
0
25
10
0.0076
0.0152
10
0
26
20
0.0304
0.0608
24
4
27
16
0.0184
0.0368
17
1
28
18
0.0208
0.0416
18
0
29
14
0.0134
0.0267
13
1
30
30
0.0329
0.0658
26
4
31
15
0.0155
0.0311
15
0
32
12
0.0114
0.0227
12
0
33
19
0.0267
0.0534
22
3
34
14
0.0140
0.0281
14
0
35
17
0.0194
0.0387
17
0
36
14
0.0137
0.0273
14
0
37
12
0.0108
0.0215
12
0
38
12
0.0111
0.0222
12
0
39
17
0.0186
0.0372
17
0
40
14
0.0138
0.0277
14
0
41
12
0.0108
0.0216
12
0
42
13
0.0113
0.0227
12
1
43
18
0.0216
0.0432
19
1
44
16
0.0176
0.0351
16
0
45
10
0.0067
0.0134
9
1
46
14
0.0148
0.0296
14
0
47
16
0.0157
0.0315
15
1
48
14
0.0128
0.0256
13
1
49
15
0.0168
0.0336
16
1
50
12
0.0110
0.0221
12
0
Table 2. Comparison of the actual and calculated age among the validation group (39 upper first premolars). Number
Actual age (years)
D/L
Calculated age (years)
Error (years)
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 37 38 39
15 14 11 17 15 14 10 10 11 11 11 13 14 30 10 15 11 31 13 10 20 16 18 14 30 15 12 19 14 17 14 12 12 17 14 12 13 18 16
0.016 0.014 0.009 0.020 0.017 0.015 0.005 0.006 0.009 0.010 0.010 0.012 0.015 0.035 0.008 0.017 0.011 0.044 0.013 0.008 0.030 0.018 0.021 0.013 0.033 0.016 0.011 0.027 0.014 0.019 0.014 0.011 0.011 0.019 0.014 0.011 0.011 0.022 0.018
15 14 11 17 16 15 8 9 11 11 11 13 15 27 10 16 12 33 13 10 24 17 18 13 26 15 12 22 14 17 14 12 12 17 14 12 12 19 16
0 0 0 0 1 1 2 1 0 0 0 0 1 3 0 1 1 2 0 0 4 1 0 1 4 0 0 3 0 0 0 0 0 0 0 0 1 1 0
estimated age when the proposed formula was applied to the validation group. The standard error of estimation, calculated by descriptive analysis, was 1.12.
Discussion Estimation of age in living persons, dead bodies, or skeletal remains is of great importance in forensic sciences. This is notably evident in investigations of mass disasters, murder inquiries of badly putrefied bodies,
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35 Actual age 30
Esmated age
Number of teeth
Error (years)
23 10 2 2 2
0 1 2 3 3
Age
25 20 15 10 5 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39
Subject's # (ascending order)
Figure 3. Comparison of the true and calculated age for the validation group.
or the examination of decomposed remains recovered from mass graves e.g. missing Kuwaiti war victims. AAR in teeth is established now as an accurate method for age estimation.1,7,10 The technique has been applied for forensic purposes over the past few decades.8,34 HPLC has been considered an easier and more convenient method of separating Asp compared to GC.8,26,29 The current study has demonstrated that HPLC technique can be effectively employed in calculating chronological age by AAR and is able to provide reliable results. Our findings have established a reasonably significant correlation of the D-/L-Asp ratio with age in a test group of 50 Kuwaiti subjects (r ¼ 0.97). Our results correspond well with previous studies utilizing HPLC techniques to determine AAR in dintin (Fu et al.: 28 first premolars, age 14–69 years, r ¼ 0.988726; Mornstad et al.: 51 various teeth, age 3– 86 years, r ¼ 0.9735; and Benesova´ et al.: 9 cases, age 18–84 years, r ¼ 0.98229). Our results are also comparable to those studies applying GC techniques (Helfman and Bada: 19 various teeth, age 5–72 years, r ¼ 0.9236; Arnany: 24 premolars, age 13–88 years, r ¼ 0.9837; Ritz et al.: 70 third molars, r ¼ 0.9938; Ohtani: 40 deciduous teeth, age 1–15 years, r ¼ 0.91432; Ohtani and Yamamoto: 56 various teeth, age 58–88 years, r ¼ 0.96114; and Ohtani and Yamamoto: 29 various teeth, age 13–70 years, r ¼ 0.984–0.99113). In our series, a linear regression line between AAR and age was recognized and could be effectively employed to estimate the individual’s age within an error range of 1.26 years (Figure 2). However, there were three cases (aged 20, 22, and 31 years) which were noticeably out of the range of the linear regression line. Since the number of subjects over 20 years of age in our series is very small (only four cases), it is not clear whether such a finding is due to the older age, or because of technical limitations on the purity of the dentin samples. Previous researchers have suggested that greater age can lead to greater errors and hence determination of age is more accurate in teeth younger than 35 years.26,39
The ultimate objective of any estimation method is to minimize the error margin between the actual age and the calculated age. In the current study, the estimated age was equal to the authentic chronological age in 26 out of 50 ‘‘test group’’ subjects (52%), with no errors of more than 4 years (Table 1). The present study has proposed an apparently reliable formula for calculating the age through AAR in Kuwaiti populations. When the proposed formula was applied to a ‘‘validation group’’ of 39 subjects, the range of error was within 1 year in 82.1% of the cases and the standard error of estimation was 1.12 years (Figure 3). The age range in this series is not large (10–31 years in the test group and 10–30 years in the validation group), due to the exclusion of carious teeth. Consequently, such a limited range may cast doubts on the validity of the proposed formula in this study when applied to older age groups. Furthermore, other studies, which have used wider age ranges, demonstrated marginally better linear associations than in this study.13,26 The type of tooth chosen for analysis may influence the results of estimation of age at death, since dentinal age is not equivalent to chronological age. It is generally agreed that the formation of permanent dentin follows the pattern of growth. However, previous studies have suggested that in elderly individuals, racemization in teeth situated deep in the oral cavity for a long time is more influenced by the environment than by the period of tooth formation.14 Moreover, earlier reports have shown that the racemization rate varies in different regions of dentin.7,9,11,40–42 It has been recommended to select the incisors or premolars for the estimation of age, as both are single-rooted, small in size, and maximum dentin is easily attainable, compared to multiple-rooted molars.13,16,25,43 The current work has employed the racemization method to the root dentin in a fairly large series of 89 first premolars. Tooth dentin is considered as one of the best target tissues10 and dentinal root has been found to give a faster rate than crowns and can provide good age estimates.10,35,38 Ohtani et al.2 has compared AAR in cementum, enamel, and dentine from first premolar teeth of the same individuals. The correlation of the ratio of D-/L-Asp with actual age was highest for dentine (r ¼ 0.992), followed by cementum (r ¼ 0.988) and enamel (r ¼ 0.961). As dentin forms from the crown towards the root apex in a series of
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concentric cones over a number of years, the D/L ratio is expected to be higher in the crown than in the root dentin among younger individuals, whereas in older teeth the reverse is true.35 Meanwhile, it has been suggested that in the elderly, the environmental temperature around the root apex might affect the degree of racemization, as a prolonged period of time has elapsed since the root formation.10 In the current study, the apical 1/3 of the root was removed in order to minimize the effects of age and bacterial infection on racemization rate.44,45 In addition to being younger than the dentinal crown, the root dentin is also much less likely to be affected by dental disease, formation of reparative dentin, and/ or dental treatment.7 In forensic practice, the crown dentin can be largely destroyed or removed (due to caries, large dental fillings, crowns, etc.), whereas the root dentin is often intact. In such instances, it is neither possible to obtain the crown dentin nor the ‘‘entire dentin of central longitudinal sections’’ – as suggested by other researchers – in a reproducible manner.40 In the meantime, numerous studies have compared racemization in different regions of the crown and root dentin,9,11,15,26,38,41,46–52 and a number of reports have demonstrated different rates of AAR in crown and root dentin within the same tooth.51,52 Recent reports have employed a whole tooth as a racemization material, with the advantage of eliminating the process of dentin isolation and standardizing the site of dentin sampling which can affect the rate of racemization.53 However, the correlation coefficient of racemization rates of the whole-tooth samples was slightly weaker than in the dentin samples. It is noteworthy to add that a study by Griffin et al.39 using HPLC has demonstrated a good correlation between ageing and AAR in teeth enamel (r ¼ 0.92). As indicated earlier, the racemization rate is strongly affected by temperature,8,54,55 and the deeper the tooth is located in the oral cavity, the higher the rate of racemization, because of higher ambient temperatures.14 In addition, the ambient temperature of the crown is likely to be different from that of the root, resulting in different racemization rates. Breathing, talking, sipping cold or hot drinks, etc. may contribute further to such thermal differences.56 The actual magnitude of such effects is unknown, but small fluctuations in temperature may bring about significant changes in the rate of racemization. Although the incisors and premolars have been recommended for this technique,16,25,43 the first premolar has been employed in the current study in order to minimize the effect of thermal variations on the racemization rate. The method described in the present study can be applied in forensic cases, even with long post-mortem intervals, since the process of racemization is almost entirely halted by death, owing to the rapid decrease in body temperature, without noteworthy
postmortem racemization.9,43 Although the majority of forensic work entails the investigation on dead bodies or remains for the purpose of age estimation, the described method necessitates the extraction of the whole analyzed tooth, and therefore is not practically applicable on living subjects. The current study is the first of its kind to investigate the correlation of AAR and age estimation in the Middle East. Recent reports have investigated differences in the racemization reaction velocities between different ethnic groups and concluded that the type of tooth selected is more important for age evaluation by racemization than ethnic variations.13 However, other additional information about possible differences within various ethnic groups is not available at the moment.34,53 Therefore, further research is required in this area in order to find out whether our findings and proposed formula can be applied to other populations. Meanwhile, it is noteworthy to add that age estimation by AAR from teeth needs specific technical requirements with a great deal of standardization which, in general, are not available in all forensic laboratories and thus cannot be employed as a routine tool for age estimation, particularly in the developing countries. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Declaration of conflicting interests The authors declare that they do not have any conflict of interest.
References 1. Ritz-Timme S, Cattaneo C, Collins MJ, et al. Age estimation: The state of the art in relation to the specific demands of forensic practice. Int J Legal Med 2000; 113: 129–136. 2. Ohtani S, Sugimoto H, Sugeno H, et al. Racemization of aspartic acid in human cementum with age. Arch oral Biol 1995; 40: 91–95. 3. Sopher IM. Forensic dentistry. Springfield, IL: Charles C Thomas, 1976, pp.113–24. 4. Valenzuela A, Martin-de las Heras S, Mandojana JM, et al. Multiple regression models for age estimation by assessment of morphologic dental changes according to teeth source. Am J Forensic Med Path 2002; 23: 386–389. 5. Pilin A, Cabala R, Pudil R, et al. The use of the D-, Laspartic ratio in decalcified collagen from human dentin as an estimator of human age. J Forensic Sci 2001; 46: 1228–1231. 6. Ball J. A critique of age estimation using attrition as the sole indicator. J Forensic Odontostomatol 2002; 20: 38–42. 7. Waite ER, Collinsa MJ, Ritz-Timmeb S, et al. A review of the methodological aspects of aspartic acid racemization analysis for use in forensic science. Forensic Sci Int 1999; 103: 113–124.
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8. Griffin RC, Penkman KEH, Moody H, et al. The impact of random natural variability on aspartic acid racemization ratios in enamel from different types of human teeth. Forensic Sci Int 2010; 200: 148–152. 9. Ogino T, Ogino H and Nagy B. Application of asparticacid racemization to forensic odontology- postmortem designation of age at death. Forensic Sci Int 1985; 29: 259–267. 10. Ohtani S and Yamamoto T. Strategy for the estimation of chronological age using the aspartic acid racemization method with special reference to coefficient of correlation between D/L ratios and ages. J Forensic Sci 2005; 50: 1020–1027. 11. Ohtani S. Estimation of age from the teeth of unidentified corpses using the amino-acid racemization method with reference to actual cases. Am J Forensic Med Pathol 1995; 16: 238–242. 12. Alkass K, Buchholz BA, Ohtani S, et al. Spalding, Age estimation in forensic sciences: Application of combined aspartic acid racemization and radiocarbon analysis. Mol Cell Proteomics 2010; 9: 1022–1030. 13. Ohtani S, Ito R and Yamamoto T. Comparison of age estimation in Japanese and Scandinavian teeth using amino acid racemization. J Forensic Sci 2011; 56: 244–247. 14. Ohtani S, Ito R and Yamamoto T. 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 2003; 117: 149–152. 15. Ogino T and Ogino H. Application to forensic odontology of aspartic acid racemization in unerupted and supernumerary teeth. J Dent Res 1988; 67: 1319–1322. 16. Carolan VA, Gardner MLG, Lucy D, et al. Some considerations regarding the use of amino acid racemization in human dentine as an indicator of age at death. J Forensic Sci 1997; 42: 10–16. 17. Ohtani S, Matsushima Y, Kobayashi Y, et al. Age estimation by measuring the racemization of aspartic acid from total amino acid content of several types of bone and rib cartilage: a preliminary account. J Forensic Sci 2002; 47: 32–36. 18. Fujii N, Ishibashi Y, Satoh K, et al. Simultaneous racemization and isomerization at specific aspartic acid residues in aB-crystallin from the aged human lens. Biochem Biophys Acta 1994; 1204: 157–163. 19. Man EH, Sandhouse ME, Burg J, et al. Accumulation of D-aspartic acid with age in the human brain. Science 1983; 220: 1407–1408. 20. Pfeiffer H, Mornstad H and Teivens A. Estimation of chronologic age using the aspartic acid racemization method. I. On human rib cartilage. Int J Legal Med 1995; 108: 19–23. 21. Pfeiffer H, Mornstad H and Teivens A. Estimation of chronologic age using the aspartic acid racemization method. II. On human cortical bone. Int J Legal Med 1995; 108: 24–26. 22. Dobberstein RC, Tung SM and Ritz-Timme S. Aspartic acid racemisation in purified elastin from arteries as basis for age estimation. Int J Legal Med 2010; 124: 269–275. 23. Kiran KK. Dental age estimation using amino acid racemization. Indian J Dent Res 2008; 19: 172–174.
24. Ohtani S, Ohhira H, Watanabe A, et al. Estimation of age from teeth by amino acid racemization: influence of fixative. J Forensic Sci 1997; 42: 137–139. 25. Yekkala R, Meers C, Van Schepdael A, et al. Racemization of aspartic acid from human dentin in the estimation of chronological age. Forensic Sci Int 2006; 15: 89–94. 26. Fu SJ, Fan CC, Song HW, et al. Age estimation using a modified HPLC determination of ratio of aspartic acid in dentin. Forensic Sci Int 1995; 73: 35–40. 27. Al-Shammari KF, Al-Khabbaz AK, Al-Ansari JM, et al. Risk indicators for tooth loss due to periodontal disease. J Periodontol 2005; 76: 1910–1918. 28. French D and Edsall JT. The reactions of formaldehyde with amino acids and proteins. Adv Protein Chem 1945; 2: 277–335. 29. Benesova T, Ales H, Pilin A, et al. A modified HPLC method for the determination of aspartic acid racemization in collagen from human dentin and its comparison with GC. J Sep Sci 2004; 27: 330–334. 30. Nimura N and Kinoshita T. Ortho-phthalaldehyde-Nacetyl-L-cysteine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid enantiomers and its applications to conventional amino acid analysis. J Chromatogr 1986; 352: 169–177. 31. Aswad DW. Determination of D- and L-aspartate in amino acid mixtures by highperformance liquid chromatography after derivatization with a chiral adduct of ophthaldialdehyde. Anal Biochem 1984; 137: 405–409. 32. Ohtani S. Age estimation by aspartic acid racemization in dentin of deciduous teeth. Forensic Sci Int 1994; 68: 77–82. 33. Hare PE, Hocrong TC and Kingjv K. Biogeochemistry of Amino Acids. New York: John Wiley and Sons, 1980, pp.503–525. 34. Arany S, Ohtani S, Yoshioka N, et al. Age estimation from aspartic acid racemization of root dentin by internal standard method. Forensic Sci Int 2004; 141: 127–130. 35. Mornstad H, Pfeiffer H and Teivens A. Estimation of dental age using HPLC-technique to determine the degree of aspartic acid racemization. J Forensic Sci 1994; 39: 1425–1431. 36. Helfman PM and Bada JL. Aspartic acid racemization in tooth enamel from living humans. Proc Natl Acad Sci USA 1975; 72: 2891–2894. 37. Arany S and Ohtani S. Age estimation by racemization method in teeth: Application of aspartic acid, glutamate, and alanine. J Forensic Sci 2010; 55: 701–705. 38. Ritz S, Schutz HW and Peper C. Postmortem estimation of age at death based on aspartic acid racemization in dentin: its applicability for root dentin. Int J Leg Med 1993; 105: 289–293. 39. Griffin RC, Moody H, Penkman KEH, et al. 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 2008; 175: 11–16. 40. Ohtani S. Different racemization ratios in dentin from different locations within a tooth. Growth Dev Aging 1997; 61: 93–9930. 41. Helfman PM and Bada JL. Aspartic acid racemization in dentin as a measure of aging. Nature 1976; 262: 279–281.
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42. Saleh N, Deutsch D and Gil-Av E. Racemization of aspartic acid in the extracellular matrix proteins of primary and secondary dentin. Calcif Tissue Int 1993; 53: 103–110. 43. Ohtani S and Yamamoto T. Age estimation by amino acid racemization in human teeth. J Forensic Sci 2010; 55: 1630–33. 44. Child AM, Gillard RD and Pollard AM. Microbially induced promotion of amino acid racemization in bone: isolation of the amino organism and detection of their enzymes. J Archaeol Sci 1993; 20: 159–168. 45. Child AM. Towards an understanding of the microbial decomposition of archaeological bone in the burial environment. J Arch Sci 1995; 22: 165–174. 46. Helfman PM and Bada JL. Aspartic acid racemization in dentin as a measure of aging. Nature 1976; 262: 279–281. 47. Ohtani S and Yamamoto K. Estimating age through the amino acid racemization of acidsoluble dentinal peptides. Jpn J Leg Med 1990; 44: 342–345. 48. Ohtani S and Yamamoto K. Estimation of age from a tooth by means of racemization of an amino-acid, especially aspartic-acid-comparison of enamel and dentin. J Forensic Sci 1992; 37: 1061–1067. 49. Shimoyama A and Harada K. An age determination of an ancient burial mound man by apparent racemization
50.
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reaction of aspartic acid in tooth dentine. Chem Lett 1984; 10: 1661–1664. Ritz S, Stock R, Schutz H-W, et al. Age estimation in biopsy specimens of dentin. Int J Leg Med 1995; 108: 135–139. Ohtani S and Yamamoto K. Age estimation using the racemization of amino acid in human dentin. J Forensic Sci 1991; 36: 792–800. Ohtani S and Yamamoto K. Age estimation using the racemization of aspartic acid in human dentin. Jpn J Leg Med 1987; 41: 181–190. Ayaka Sakuma, Susumu Ohtani, Hisako Saitoh, et al. Comparative analysis of aspartic acid racemization methods using whole-tooth and dentin samples. Forensic Sci Int 2012; 223: 198–201. Schroeder RA and Bada JL. Glacial-postglacial temperature difference deduced from aspartic acid racemization in fossil bones. Science 1973; 182: 479–482. Ohtani S. Technical notes for age estimation using the femur: influence of various analytical conditions on Daspartic acid contents. Int J Leg Med 2002; 116: 361–364. Brown AC and Goldberg MP. Surface temperatures and temperature gradient in human teeth. Oral Biol 1966; 11: 973–982.
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Med Sci Law OnlineFirst, published on March 3, 2014 as doi:10.1177/0025802414524383
Original article
Racemization of aspartic acid in root dentin as a tool for age estimation in a Kuwaiti population
Medicine, Science and the Law 0(0) 1–8 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0025802414524383 msl.sagepub.com
Mohamed Amin Elfawal1, Sahib Issa Alqattan2 and Noha Ayman Ghallab3
Abstract Estimation of age is one of the most significant tasks in forensic practice. Amino acid racemization is considered one of the most reliable and accurate methods of age estimation and aspartic acid shows a high racemization reaction rate. The present study has investigated the application of aspartic acid racemization in age estimation in a Kuwaiti population using root dentin from a total of 89 upper first premolar teeth. The D/L ratio of aspartic acid was obtained by HPLC technique in a test group of 50 subjects and a linear regression line was established between aspartic acid racemization and age. The correlation coefficient (r) was 0.97, and the standard error of estimation was 1.26 years. The racemization age ‘‘t’’ of each subject was calculated by applying the following formula: ln [(1 þ D/L)/(1 D/L)] ¼ 0.003181 t þ (0.01591). When the proposed formula ‘‘estimated age t ¼ ln [(1 þ D/L)/(1 D/L)] þ 0.01591/0.003181’’ was applied to a validation group of 39 subjects, the range of error was less than one year in 82.1% of the cases and the standard error of estimation was 1.12. The current work has established a reasonably significant correlation of the D-/L-aspartic acid ratio with age, and proposed an apparently reliable formula for calculating the age in Kuwaiti populations through aspartic acid racemization. Further research is required to find out whether similar findings are applicable to other ethnic populations. Keywords age estimation, aspartic acid, racemization, teeth
Introduction Identification is one of the important functions in forensic work. Among establishing many aspects of identification of a human body, estimation of age has imperative significance. Age estimation in living subjects, cadavers, and human remains may elucidate matters with considerable legal and social consequences. The implications of a ‘‘lack of identification’’ of a cadaver cannot be underrated. The number of unidentified cadavers and human remains throughout the world is on the increase.1 Teeth, the hardest of all human tissues, are highly resistant to physical and chemical influences. Even in cases of extensive destruction of the body, the teeth are often preserved, especially the molars.2 Meanwhile, there are legal circumstances requiring age estimation in living individuals without valid proof of date of birth e.g. adolescent drug smugglers in Asian and Gulf countries. Determination of age using teeth have been studied by numerous workers and updated with new techniques, ranging from conventional morphological methods3,4 to biochemical analytical techniques.5
Age estimation in children and adolescents frequently depends on radiological examination of skeletal and dental development. In adults, however, such traditional methods for ageing are highly subjective.6–8 Conversely, aspartic acid racemization (AAR) is considered one of the most reliable and accurate methods of age estimation in forensic practice.8–13 Racemization is a chemical reaction in which spontaneous conversion of L-enantiomer into D-enantiomer occurs in the course of ageing and aspartic acid (Asp) shows a high racemization reaction rate.14 Throughout time, such L-amino acids are partly converted to D-enantiomers until an equilibrium mixture of both is attained.15 Consequently, measurement of 1
Faculty of Medicine, Kuwait University, Saffat, Kuwait Ministry of Interior Kuwait, Kuwait 3 Faculty of Oral and Dental Medicine, Cairo University, Cairo, Egypt 2
Corresponding author: Mohamed Amin Elfawal, Faculty of Medicine, Kuwait University, Saffat, Kuwait, 13110 Kuwait. Email: [email protected]
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the D:L Asp ratio will give an indication of the time elapsed since the dentin was laid down. Racemization takes place in tissues with slow metabolism, such as teeth, bones, cartilages, eye-lens, or brain.16–19 Nevertheless, employing non-dental tissues, e.g. cartilage, bone, or elastin, in age estimation by AAR has shown a less significant correlation compared to dentinal proteins.20–22 The inter-conversion of Asp enantiomers is a reversible first-order chemical reaction. It is influenced by several factors such as type of tooth, analyzed, tooth part, surrounding temperature, humidity, pH, and fixative used.23,24 It has been found that the earlier the tooth formation is accomplished, the higher the level of D-Asp. Accordingly, different types of teeth of the same individual, formed throughout different periods of growth, would have varied levels of D-Asp.14 Hence, a specific type of tooth is required to be employed as a standard reference and is expected to yield better results than the use of various types of teeth for age estimation in medico-legal practice.25 In the present study, we have investigated the application of AAR in root dentin from upper first premolar teeth as a tool in age estimation in a Kuwaiti population.
Materials and methods Chemicals Powders of D- and L-aspartic acid, O-phthaldialdehyde (OPA), N-acetyl-L-cysteine (NAC), phenylmethyl sulfonyl fluoride, and alpha toluene sulfonel fluoride were all purchased from Sigma-Aldrich (USA).
Reagent preparation OPA / NAC reagent was prepared by dissolving OPA (8 mg) in methanol (600 mL), and then 500 mL of 0.4 M Na borate buffer (pH 9.4) and 120 mL of 1 M NAC were added. The reagent stock solution was stored at 4 C.
Instrumentation and operating procedures The HPLC system (Waters, Milford, Massachusetts, USA) consisted of 600S Controller, 626 Pump Pressure, 717 Plus Autosampler and 474 Scanning Fluorescence Detector. The fluorescence detection was used at ex 340 nm and em 420 nm. The pump pressure was maintained at 1800 psi. Research analytical oven was used for hydrolysis purposes (Oldham, Lancs, England). A Kromasil C8, 5 mm chiral column (4.6 250 mm) (USA) was used for separation of D- and L-Asp. A gradient elution was carried out with two solutions: A and B. Solution A was made of 50 mM sodium acetate pH 5.9 and solution B was made of 80% methanol and 20% of
solution A. During the first 5 minutes the elution was 90% of solution A and 10% of solution B, then the percentage of solution B was increased up to 100% linearly over a period of 5 min. The flow rate of the mobile phase was 1 mL/min and the injection volume was 10 mL.
Sample preparation and derivatization The method used in this work was similar to those employed by Yekkala et al.25 and Fu et al.26 A total of 89 upper first premolar teeth were collected. They were divided into two groups, a test group included 50 teeth (33 males and 17 females) of known age (age range between 10 and 31 years and mean age 14.98 4.76), and a validation group consisted of 39 teeth (26 males and 13 females) of recorded age, but unknown to the investigator (age range between 10 and 30 years and mean age 15.1 5.16). All teeth studied were sound, free from restorations and dental caries, and were extracted from living Kuwaiti individuals because of periodontal diseases or orthodontic reasons. Recent reports have shown that periodontal disease is the commonest underlying cause of teeth extraction in Kuwaiti adults.27 Teeth were stored frozen-dry to avoid the influence of fixatives.21,28 The crown and apical 1/3 of the root were separated using dental diamond disc and bur, both cooled in liquid nitrogen in order to avoid heatrelated racemization of Asp. The dentin samples were broken into small shards, crushed into powder, and 40–80 mg were washed in 15% NaCl at 4 C overnight, centrifuged (13000 r/min for 5 min) three times, and dried by a cold system model and a high vacuum pump. The dry residue was re-suspended in 1 mL of 6 M HCl and hydrolyzed for 6 hours. The hydrolyzates were dried by the same vacuum pump apparatus, desalted on a cation exchange column, and 1 mL of double distilled water was added. The dentin samples which contained the amino acids were passed down the column. Finally, 6 mL of 2 M ammonium hydroxide solution were used to elute the amino acids which were then collected in a clean glass tube. Four of 1.5 mL aliquots were made and then derivatized.29–31 One of the four aliquots was reconstituted with double distilled water for derivatization and the other three were stored for further analysis. Derivatization was accomplished by mixing 10 mL of the prepared sample solution with 20 mL of OPANAC derivatization reagent. After 2.5 minutes, 200 mL of 50 mmol Na acetate was added, and then 10 mL of the derivatized solution was injected into the HPLC system. The HPLC analysis was done under ambient temperature of 23 C. The total run time was 14 minutes and the retention time for D-Asp was 9.767 minutes and that for L-Asp was 11.683 minutes (Figure 1). A control was made using water, instead of the dentin sample, under the same conditions and gave no reaction.
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The analysis of the D/L ratio for Asp in the dentin samples was calculated. The formula ln [(1 þ D/L) / (1 D/L)] was employed.32 The estimated age was assessed from the D/L ratio based on the fact that racemization of Asp follows a first order reversible rate law equation: ln [(1 þ D/L) / (1 D/ L)] ¼ 2kt þ A,33 where D/L is the ratio of the area of eluted peaks of D and L Asp, 2k is the constant ‘‘slope’’ of racemization, which can be calculated from a linear regression of ln(1þD/L) / (1-D/L)] versus age of the individual, t is the estimated age (years), and A is the intercept of the linear regression line with the Y axis.
investigator. According to the proposed formula, the age was calculated as follows: Estimated age t ¼
ln ½ð1 þ D=LÞ=ð1 D=LÞ þ 0:01591 0:003181
Table 2 and Figure 3 illustrate the comparison between the actual and calculated age, employing the proposed formula, among the validation group. Table 3 shows the distribution of error values in the
0.1 Series1
Results
0.09
Linear (Series1)
0.08
ln [(1+D/L) / (1-D/L)]
The root dentin of 50 upper first premolars (test group) was analyzed and the D/L ratio of Asp was obtained. A linear regression line was established, which is a plot of ln [(1 þ D/L) / (1 D/L)] of Asp versus the known age (Figure 2). It was used to calculate the 2k and A values for the kinetic equation of racemization of amino acids: ln [(1 þ D/L) / (1 D/ L)] ¼ 2kt þ A. Accordingly, the racemization age ‘‘t’’ of each subject was calculated by applying the following formula: ln [(1 þ D/L)/(1 D/L)] ¼ 0.003181 t þ (0.01591). The correlation coefficient value (r) was 0.97 and the coefficient of determination (r2) was 0.92. The standard error of estimation, calculated by descriptive analysis, was 1.26 years (actual age minus racemization age). Table 1 shows the experimental data and racemization age for the test group. The proposed formula was applied to a validation group of 39 upper first premolars, where the age of the subject has been recorded but was unknown to the
0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0
10
20
30
40
Age (years)
Figure 2. Linear regression line for the test group (total number 50 premolars).
Figure 1. HPLC chromatogram of aspartic acid separation (C ¼ 1 mg/mL methanol).
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Table 1. Experimental data and racemization age estimation for the test group (50 upper first premolar teeth). Actual age
D/L
ln [(1 þ D/L)/ (1 D/L)]
Racemization age
1
16
0.0157
0.0315
15
1
2
14
0.0128
0.0256
13
1
3
15
0.0168
0.0336
16
1
4
12
0.0110
0.0221
12
0
5
15
0.0160
0.0320
15
0
6
14
0.0143
0.0287
14
0
7
11
0.0094
0.0188
11
0
8
17
0.0198
0.0396
17
0
9
15
0.0171
0.0343
16
1
No.
Error (years)
10
14
0.0153
0.0306
15
1
11
10
0.0050
0.0099
8
2
12
10
0.0061
0.0122
9
1
13
11
0.0091
0.0182
11
0
14
11
0.0095
0.0190
11
0
15
11
0.0098
0.0195
11
0
16
22
0.0323
0.0645
25
3
17
13
0.0120
0.0240
13
0
18
14
0.0152
0.0305
15
1
19
30
0.0350
0.0699
27
3
20
10
0.0084
0.0167
10
0
21
15
0.0172
0.0344
16
1
22
11
0.0107
0.0213
12
1
23
31
0.0444
0.0889
33
2
24
13
0.0126
0.0251
13
0
25
10
0.0076
0.0152
10
0
26
20
0.0304
0.0608
24
4
27
16
0.0184
0.0368
17
1
28
18
0.0208
0.0416
18
0
29
14
0.0134
0.0267
13
1
30
30
0.0329
0.0658
26
4
31
15
0.0155
0.0311
15
0
32
12
0.0114
0.0227
12
0
33
19
0.0267
0.0534
22
3
34
14
0.0140
0.0281
14
0
35
17
0.0194
0.0387
17
0
36
14
0.0137
0.0273
14
0
37
12
0.0108
0.0215
12
0
38
12
0.0111
0.0222
12
0
39
17
0.0186
0.0372
17
0
40
14
0.0138
0.0277
14
0
41
12
0.0108
0.0216
12
0
42
13
0.0113
0.0227
12
1
43
18
0.0216
0.0432
19
1
44
16
0.0176
0.0351
16
0
45
10
0.0067
0.0134
9
1
46
14
0.0148
0.0296
14
0
47
16
0.0157
0.0315
15
1
48
14
0.0128
0.0256
13
1
49
15
0.0168
0.0336
16
1
50
12
0.0110
0.0221
12
0
Table 2. Comparison of the actual and calculated age among the validation group (39 upper first premolars). Number
Actual age (years)
D/L
Calculated age (years)
Error (years)
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 37 38 39
15 14 11 17 15 14 10 10 11 11 11 13 14 30 10 15 11 31 13 10 20 16 18 14 30 15 12 19 14 17 14 12 12 17 14 12 13 18 16
0.016 0.014 0.009 0.020 0.017 0.015 0.005 0.006 0.009 0.010 0.010 0.012 0.015 0.035 0.008 0.017 0.011 0.044 0.013 0.008 0.030 0.018 0.021 0.013 0.033 0.016 0.011 0.027 0.014 0.019 0.014 0.011 0.011 0.019 0.014 0.011 0.011 0.022 0.018
15 14 11 17 16 15 8 9 11 11 11 13 15 27 10 16 12 33 13 10 24 17 18 13 26 15 12 22 14 17 14 12 12 17 14 12 12 19 16
0 0 0 0 1 1 2 1 0 0 0 0 1 3 0 1 1 2 0 0 4 1 0 1 4 0 0 3 0 0 0 0 0 0 0 0 1 1 0
estimated age when the proposed formula was applied to the validation group. The standard error of estimation, calculated by descriptive analysis, was 1.12.
Discussion Estimation of age in living persons, dead bodies, or skeletal remains is of great importance in forensic sciences. This is notably evident in investigations of mass disasters, murder inquiries of badly putrefied bodies,
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35 Actual age 30
Esmated age
Number of teeth
Error (years)
23 10 2 2 2
0 1 2 3 3
Age
25 20 15 10 5 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39
Subject's # (ascending order)
Figure 3. Comparison of the true and calculated age for the validation group.
or the examination of decomposed remains recovered from mass graves e.g. missing Kuwaiti war victims. AAR in teeth is established now as an accurate method for age estimation.1,7,10 The technique has been applied for forensic purposes over the past few decades.8,34 HPLC has been considered an easier and more convenient method of separating Asp compared to GC.8,26,29 The current study has demonstrated that HPLC technique can be effectively employed in calculating chronological age by AAR and is able to provide reliable results. Our findings have established a reasonably significant correlation of the D-/L-Asp ratio with age in a test group of 50 Kuwaiti subjects (r ¼ 0.97). Our results correspond well with previous studies utilizing HPLC techniques to determine AAR in dintin (Fu et al.: 28 first premolars, age 14–69 years, r ¼ 0.988726; Mornstad et al.: 51 various teeth, age 3– 86 years, r ¼ 0.9735; and Benesova´ et al.: 9 cases, age 18–84 years, r ¼ 0.98229). Our results are also comparable to those studies applying GC techniques (Helfman and Bada: 19 various teeth, age 5–72 years, r ¼ 0.9236; Arnany: 24 premolars, age 13–88 years, r ¼ 0.9837; Ritz et al.: 70 third molars, r ¼ 0.9938; Ohtani: 40 deciduous teeth, age 1–15 years, r ¼ 0.91432; Ohtani and Yamamoto: 56 various teeth, age 58–88 years, r ¼ 0.96114; and Ohtani and Yamamoto: 29 various teeth, age 13–70 years, r ¼ 0.984–0.99113). In our series, a linear regression line between AAR and age was recognized and could be effectively employed to estimate the individual’s age within an error range of 1.26 years (Figure 2). However, there were three cases (aged 20, 22, and 31 years) which were noticeably out of the range of the linear regression line. Since the number of subjects over 20 years of age in our series is very small (only four cases), it is not clear whether such a finding is due to the older age, or because of technical limitations on the purity of the dentin samples. Previous researchers have suggested that greater age can lead to greater errors and hence determination of age is more accurate in teeth younger than 35 years.26,39
The ultimate objective of any estimation method is to minimize the error margin between the actual age and the calculated age. In the current study, the estimated age was equal to the authentic chronological age in 26 out of 50 ‘‘test group’’ subjects (52%), with no errors of more than 4 years (Table 1). The present study has proposed an apparently reliable formula for calculating the age through AAR in Kuwaiti populations. When the proposed formula was applied to a ‘‘validation group’’ of 39 subjects, the range of error was within 1 year in 82.1% of the cases and the standard error of estimation was 1.12 years (Figure 3). The age range in this series is not large (10–31 years in the test group and 10–30 years in the validation group), due to the exclusion of carious teeth. Consequently, such a limited range may cast doubts on the validity of the proposed formula in this study when applied to older age groups. Furthermore, other studies, which have used wider age ranges, demonstrated marginally better linear associations than in this study.13,26 The type of tooth chosen for analysis may influence the results of estimation of age at death, since dentinal age is not equivalent to chronological age. It is generally agreed that the formation of permanent dentin follows the pattern of growth. However, previous studies have suggested that in elderly individuals, racemization in teeth situated deep in the oral cavity for a long time is more influenced by the environment than by the period of tooth formation.14 Moreover, earlier reports have shown that the racemization rate varies in different regions of dentin.7,9,11,40–42 It has been recommended to select the incisors or premolars for the estimation of age, as both are single-rooted, small in size, and maximum dentin is easily attainable, compared to multiple-rooted molars.13,16,25,43 The current work has employed the racemization method to the root dentin in a fairly large series of 89 first premolars. Tooth dentin is considered as one of the best target tissues10 and dentinal root has been found to give a faster rate than crowns and can provide good age estimates.10,35,38 Ohtani et al.2 has compared AAR in cementum, enamel, and dentine from first premolar teeth of the same individuals. The correlation of the ratio of D-/L-Asp with actual age was highest for dentine (r ¼ 0.992), followed by cementum (r ¼ 0.988) and enamel (r ¼ 0.961). As dentin forms from the crown towards the root apex in a series of
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concentric cones over a number of years, the D/L ratio is expected to be higher in the crown than in the root dentin among younger individuals, whereas in older teeth the reverse is true.35 Meanwhile, it has been suggested that in the elderly, the environmental temperature around the root apex might affect the degree of racemization, as a prolonged period of time has elapsed since the root formation.10 In the current study, the apical 1/3 of the root was removed in order to minimize the effects of age and bacterial infection on racemization rate.44,45 In addition to being younger than the dentinal crown, the root dentin is also much less likely to be affected by dental disease, formation of reparative dentin, and/ or dental treatment.7 In forensic practice, the crown dentin can be largely destroyed or removed (due to caries, large dental fillings, crowns, etc.), whereas the root dentin is often intact. In such instances, it is neither possible to obtain the crown dentin nor the ‘‘entire dentin of central longitudinal sections’’ – as suggested by other researchers – in a reproducible manner.40 In the meantime, numerous studies have compared racemization in different regions of the crown and root dentin,9,11,15,26,38,41,46–52 and a number of reports have demonstrated different rates of AAR in crown and root dentin within the same tooth.51,52 Recent reports have employed a whole tooth as a racemization material, with the advantage of eliminating the process of dentin isolation and standardizing the site of dentin sampling which can affect the rate of racemization.53 However, the correlation coefficient of racemization rates of the whole-tooth samples was slightly weaker than in the dentin samples. It is noteworthy to add that a study by Griffin et al.39 using HPLC has demonstrated a good correlation between ageing and AAR in teeth enamel (r ¼ 0.92). As indicated earlier, the racemization rate is strongly affected by temperature,8,54,55 and the deeper the tooth is located in the oral cavity, the higher the rate of racemization, because of higher ambient temperatures.14 In addition, the ambient temperature of the crown is likely to be different from that of the root, resulting in different racemization rates. Breathing, talking, sipping cold or hot drinks, etc. may contribute further to such thermal differences.56 The actual magnitude of such effects is unknown, but small fluctuations in temperature may bring about significant changes in the rate of racemization. Although the incisors and premolars have been recommended for this technique,16,25,43 the first premolar has been employed in the current study in order to minimize the effect of thermal variations on the racemization rate. The method described in the present study can be applied in forensic cases, even with long post-mortem intervals, since the process of racemization is almost entirely halted by death, owing to the rapid decrease in body temperature, without noteworthy
postmortem racemization.9,43 Although the majority of forensic work entails the investigation on dead bodies or remains for the purpose of age estimation, the described method necessitates the extraction of the whole analyzed tooth, and therefore is not practically applicable on living subjects. The current study is the first of its kind to investigate the correlation of AAR and age estimation in the Middle East. Recent reports have investigated differences in the racemization reaction velocities between different ethnic groups and concluded that the type of tooth selected is more important for age evaluation by racemization than ethnic variations.13 However, other additional information about possible differences within various ethnic groups is not available at the moment.34,53 Therefore, further research is required in this area in order to find out whether our findings and proposed formula can be applied to other populations. Meanwhile, it is noteworthy to add that age estimation by AAR from teeth needs specific technical requirements with a great deal of standardization which, in general, are not available in all forensic laboratories and thus cannot be employed as a routine tool for age estimation, particularly in the developing countries. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Declaration of conflicting interests The authors declare that they do not have any conflict of interest.
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