Assessment of Dental Age Estimation Using Aspartic Acid Racemization in Human Dentin in Single-Rooted Teeth

Neelima Dasarathi; Kiran Kumar Kattappagari; Venkata Suresh Ponnuru; Ravi Teja Chitturi Suryaprakash; Arvind Babu Santhosh; Baddam Venkat Ramana Reddy

doi:10.4103/jofs.jofs_90_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 14 | Issue : 2 | Page : 114-119

Assessment of Dental Age Estimation Using Aspartic Acid Racemization in Human Dentin in Single-Rooted Teeth

Neelima Dasarathi¹, Kiran Kumar Kattappagari², Venkata Suresh Ponnuru³, Ravi Teja Chitturi Suryaprakash⁴, Arvind Babu Santhosh⁵, Baddam Venkat Ramana Reddy²
¹ Neelima Dental Clinic, Guntur, Andhra Pradesh, India
² Department of Oral Pathology, SIBAR Institute of Dental Sciences, Takkellapadu, Guntur, Andhra Pradesh, India
³ Department of Pharmaceutical Sciences, Krishna University, Machilipatnam, Andhra Pradesh, India
⁴ UWA Dental School, 17 Monash Ave, Needlands, WA, Australia
⁵ Faculty of Medical Sciences, Univeristy of West Indies, Mona, Jamaica, West Indies

Date of Submission	22-Mar-2022
Date of Decision	07-Oct-2022
Date of Acceptance	20-Oct-2022
Date of Web Publication	10-Jan-2023

Correspondence Address:
Kiran Kumar Kattappagari
Department of Oral Pathology, SIBAR Institute of Dental Sciences, Takkellapadu, Guntur – 566009, Andhra Pradesh
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jofs.jofs_90_22

Abstract

Introduction: Dental age estimation has always been a very important and very susceptible subject in forensic science, as it is an essential requirement in living, dead human individual’s remains. Aspartic acid racemization is considered to be one of the advanced, reliable, accurate, and complex biochemical methods. There will be an amendment of Levo aspartic acid to dexto aspartic acid when the age advances. To estimate the dental age of single-rooted teeth using aspartic acid racemization. Material and Methods: Thirty anterior teeth were collected from maxillary and mandibular arches distributed into four groups. All teeth were analyzed for Levo (l) and Dextro (d) form of aspartic acid using a high-performance liquid chromatography technique and the ratio of amino acid racemization for each sample was determined. The obtained data were subjected to step-wise linear regression analysis for the ratio of d and l aspartic acid against actual age with estimated age. Results: Distribution of teeth about age maximum samples was collected at 51 to 60 years. l-aspartic acid ratio was more in the younger age group but when the age increases d-aspartic acid ratio was increased. Linear regression analysis showed maximum samples showed very close to the actual age (30%). Conclusion: By analyzing the levels of aspartic acid using amino acid racemization, we can estimate an individual with maximum accuracy. Accretion of d-aspartic acid in dentin is synchronous with the aging of an individual and this method can be used as one of the precise methods for dental age estimation.

Keywords: Aspartic acid, dental age, dentin, forensic odontology

How to cite this article:
Dasarathi N, Kattappagari KK, Ponnuru VS, Suryaprakash RT, Santhosh AB, Reddy BR. Assessment of Dental Age Estimation Using Aspartic Acid Racemization in Human Dentin in Single-Rooted Teeth. J Orofac Sci 2022;14:114-9

How to cite this URL:
Dasarathi N, Kattappagari KK, Ponnuru VS, Suryaprakash RT, Santhosh AB, Reddy BR. Assessment of Dental Age Estimation Using Aspartic Acid Racemization in Human Dentin in Single-Rooted Teeth. J Orofac Sci [serial online] 2022 [cited 2023 Jun 9];14:114-9. Available from: https://www.jofs.in/text.asp?2022/14/2/114/367450

Introduction

Dental age estimation is an important tool utilized for specifically identifying dead human individual remains.^[1] In living individuals, age estimation is used in various settings/scenarios such as in identifying criminals for legal proceedings, social requirements; namely, for birth, marriage records. Age assessment is mandatory for logistic purposes for individuals seeking employment in the government offices, the armed forces, and age determines the retirement age of employees in government and private sectors. Other than these aspects such as personal identification of human remains due to natural and manmade disaster.^[2] The assignment of age is, therefore, a crucial process and its estimation thereof should be as accurate as possible.^[3] The estimation of chronological age has been performed by various methods such as morphological, histological, radiographically, and biochemical methods.^[4] Chronological age of living and deceased human beings has been estimated using morphological, histological, radiographic, biochemical, and molecular methods. If deceased body is in good condition the apparent age can be estimated visually. But in most situations, the bodily remains are completely or partially degraded making the visual and morphological identification difficult. Teeth and the supporting bone are often preserved even when the rest of the body is severely damaged as they are protected by the surrounding soft tissues. This facilitates the use of histological and biochemical techniques to identify the teeth and estimate the age.^[5],[6] Among the current methods, aspartic acid racemization process and its quantitative estimation considered to be an accurate indicator of dental age as it is reported to have an accuracy rate of approximately ±3 years.^[7] Whereas analysis based on morphological methods is reported to yield an error margin ±10 years. Racemization is the natural process that will eventually convert one enantiomer of a compound, such as l-amino acid, converts to the other enantiomer, that is, d-amino acid. Aspartic acid racemization is considered to be one of the advanced, reliable, accurate, and complex biochemical methods.^[8]

As tooth is one of the strongest structures in the human body, it can withstand climatic and thermal changes. There are many changes that occur in proteins such as isomerization, oxidation, and racemization. Among all these changes racemization is the chemical reaction from l-amino acids to d-amino acids and its correlating highly with protein age.^[9] In the living body, newly synthesized proteins are normally made synthesized l-form amino acids, although there are some exceptional peptides that are biologically synthesized using d-form amino acids.^[10] l-form amino acids within protein changes to d-form by a self-modulated chemical reaction. This conversion of l-form to d-form influenced by some of factors such as temperature, humidity, and pH values of 8. Estimating the chronological and dental age is reported to be more accurate in organs with low metabolic rate such as bone, cartilage, teeth, brain cells, and eye lens when compared to organs with high metabolic rates.^[9] Even though numerous methods have been found useful in dental age estimation, we undertook this study in single-rooted teeth to substantiate and reiterate the use of aspartic acid racemization and its quantification in routine practice of forensic odontology.

Material and Methodology

Sample size was 30 anterior teeth were collected both maxillary and mandibular arches. Sampling method was convenience sampling method. Time of study period was 8 months. The sample size calculation was done using G* power (Software. informer) 3.1 version.

Inclusion criteria: Thirty maxillary and mandibular anterior teeth were collected from individuals who reported to the out-patient department of our institution with the chief complaint of mobility of teeth due to periodontal infection. The study group divided based on age in Group I : 40 to 50 years (n = 7), Group II: 51 to 60 years (n = 14), Group III: 61 to 70 years (n = 6), and Group IV: >70 years (n = 3).

Exclusion criteria: Individuals with attrition, abrasion, erosion, and dental caries were excluded from this study. Thirty samples are divided into four groups:

Group I : 40 to 50 years (n = 7)
Group II : 51 to 60 years (n = 14)
Group III: 61 to 70 years (n = 6)
Group IV: ≥70 years (n = 3).

Ethical approval for this study (protocol no. 41/IEC/SIBAR/2014) from SIBAR institutional ethics committee, Guntur on 4th july 2014.

Samples preparation

After extraction the teeth were collected and soft tissue attachment was removed with scalpel. They were treated with hypochlorite solution and placed in sterile container contains 10% neutral buffered formalin solution to preserve the organic and inorganic components. Later they were immersed in a solution of 5% sodium hypo chloride for 30 minutes following this tooth they were washed under running tap water and air dried. Each tooth was cut bucco lingually at enamel and cementum surface using diamond bur with air rotor hand piece with continuous waster hand piece jet spray to prevent alterations of amino acid structure. The dentin was stored in a sealed container and labeled with the sample number, date, and actual age of those particular individuals. Analysis of aspartic acid racemization was performed using the criteria described by Ohtani and Yamamoto in 2005.^[9]

Equipment used for aspartic acid estimation

High Performance Liquid Chromatography (HPLC) with auto sampler collection (Agilent LC 1220 infinity, CA, USA). The ratio of d and l aspartic acid was calculated using linear regression formula for correlating the actual age with estimated age for all 30 samples Ln [(1 + d/l)/(1–d/l)].

Statistical analysis

The data obtained were subjected to linear regression analysis using SPSS statistical software package (SPSS 20.0: Inc., Chicago, IL, USA). The step wise linear regression analysis was done for the ratio of Dextro and Levo aspartic acid against actual age [Table 1].

Table 1 Summary of coefficient of correlation between D/L ratio and ages in various human structures for age estimation from aspartic acid racemization

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Results

Our study contains 30 single rooted anterior teeth. Among these 30 teeth 20 were collected from females and 10 from males. The actual chronological age of the sample ranged from 40 to 80 years. Distribution of gender accordance with different groups, in group II (30.0%) males were more followed by group I (16.6%) [Figure 1]. The type of characterization of teeth and their distribution according to groups was done. Among all the samples, lateral incisors were more in number (46.6%), followed by canines (30.0%), and central incisors (23.3%). Comparison of l (Levo) aspartic acid to d (Dextro) aspartic acid in different groups. Distribution of l-aspartic acid ratio decreased with increasing age, whereas, d-aspartic acid ratio increased in older individuals samples. The distribution of l and d ratio observed in [Figure 2]. Method of validation was done by measuring linearity of dental age with actual age, precision values in inter- and intra-observation of dental age stability for all these samples. Values of both d and l aspartic acid values were increased when concentration of aspartic acid increased [Figure 3] and [Figure 4].

Figure 1 Distribution of samples with gender in different groups.

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Figure 2 Distribution of l-aspartic acid and d-aspartic acid in different groups.

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Figure 3 Linearity values of Dextro form of aspartic acid correlation with concentration of acid.

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Figure 4 Linearity values of Levo form of aspartic acid correlation with concentration of acid.

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The ratio between d/l aspartic acid in relation to actual age with estimated age in all 30 samples. Five samples showed actual age to be similar with estimated age. The difference of age was 0.10 ± 0.62 years. The difference of age showed in [Table 2]. The linear regression analysis was done for the ratio of Dextro and Levo aspartic acid against the actual age of teeth. Maximum 0.4333 samples have shown very close results to the actual age but only two samples were shown d/l aspartic acid ratio correlating the actual age with estimated age shown in [Figure 5].

Table 2 The ratio between D/L aspartic acid in relation to actual age with estimated age in all 30 samples

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Figure 5 Age estimation using aspartic acid against of teeth in the linear regression line.

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Discussion

Growth in human beings is a continuous change and it takes place from the birth to senility.

Hard tissues like bone and teeth undergo changes with growth.^[13] As estimation of age at death is a key factor in forensic and archaeological departments. Forensic odontology plays an important role in age estimation. Few advanced technologies like DNA finger printing, amelogylphics, and restriction fragment length polymorphism (RFLP) are also in existence.^[14] All these factors are not foolproof and errors are known to occur. Aspartic acid racemization was proposed by several researchers and it has been successful in forensic age estimation with maximum accuracy. This method was developed based on the principle of racemization, which follows a reversible first order chemical reaction.^[15]

Aspartic acid racemization is an optical phenomenon in which the amino acids contain one asymmetric carbon atom in their molecules. This carbon atom shows optical activity, that is; the Dextro and Levo enantiomers of these molecules rotate the plane of polarized light to the right (Dextro) or to the left (Levo). In most of the living organisms, these optically active amino acids initially consist of only the Levo-enantiomers. As the age advances, these Levo amino acids are partly converted to the Dextro-enantiomers, until the equilibrium mixtures are attained. Several amino acids such as aspartic acid, alanine, glutamic acid, serine, isoleucine, and leucine have this property. Among all the amino acids, aspartic acid has the highest racemization rate and hence, is preferred for dental age estimation.^[16]

Ohtani^[17] considered teeth to be optimal for this racemization procedure, because teeth contain hard structures such as enamel, dentin, and cementum which could be utilized for racemization procedure. Our study utilized dentin from teeth for this purpose. The organic substance of dentin consists of acid insoluble fraction such as collagen and very little percentage of non collagenous.

Aspartic acid racemization is rapid in non-collagen proteins but proceeds slowly in collagen proteins.^[18] Since dentin consists more collagen, racemization of aspartic acid in dentin can be readily used to estimate the dental age. Dentin thickness will be more, so it will be preferred for racemization procedure. Degree of racemization will be different in different types of teeth, based on presence of dentin volume, it was suggested that single rooted teeth give an accurate value when compared to multi rooted teeth.

Among single rooted teeth, canine considered to be ideal for amino acid racemization as it has a greater surface area than the incisors and it lasts for longer periods of time in the oral cavity.^[19]

In our study we have considered anterior teeth (central incisor, lateral incisor, canines). Percentage of error was less in both canines and central incisors, followed by lateral incisors. Volume of availability of dentin was comparatively low in case of lateral incisor than the centrals and canines.

These findings are similar to Ritz et al., in 1990. According to Rajkumari et al., in 2013 the ratio of D / L in younger individuals was lesser, when the age advances the ratio of D / L/was increases.^[20] Our observations pertaining to the d/l ratio more in younger age group when compared with older age group. We used the least square method to calculate the equation of the regression analysis, the observations suggested that there is a relationship between the extent of racemization and age with respect to each tooth. It showed that the d/l aspartic acid ratio increases with the increasing age. The correlation coefficient was obtained as r = 0.9923 between the age and the ratio of d/l enantiomers of aspartic acid. This is in accordance with the studies done by Rajkumari et al., in 2013 in dentin was r = 0.9542 and Ohtani et al., in longitudinal sections of dentin r = 0.9612.^[20] The present study reinforces the fact that there is a gradual increase in the d/l ratio of the samples, in accordance with age. There was a simultaneous increase in the d-enantiomer of the aspartic acid with advancing age. Aspartic acid racemization and its correlation in age estimation, was confirmed with a very minimal mean error of ±2 years. Hence it accounts to be a very accurate and precise method for age estimation.

Conclusion

The present study concludes that there is simultaneous increase in the d-form of aspartic acid and decrease in the l-form of aspartic acid when age advanced. By analyzing the levels of aspartic acid using amino acid racemization we can estimate the age of an individual with maximum accuracy. In order to depend on the evaluated unknown ages of teeth using this method, it is essential to perform a set of experiments using known age of teeth until the standard errors are within ±3 years. However, to attain internationally acceptable results, the control teeth can be substituted by standard specimens, that are prepared from mixtures of d- and l-aspartic acids. This shows the possibility of using the standard specimens in other laboratories, so that the racemization ratio can be measured with sufficient reproducibility, which is always an issue.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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