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SHORT COMMUNICATION Table of Contents   
Year : 2008  |  Volume : 19  |  Issue : 2  |  Page : 172-174
Dental age estimation using amino acid racemization


Department of Oral and Maxillofacial Pathology, SDM College of Dental Sciences and Hospital, Dharwad - 580 009, Karnataka, India

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Date of Submission12-Dec-2006
Date of Decision05-Sep-2007
Date of Acceptance27-Oct-2007
 

How to cite this article:
Kiran Kumar K. Dental age estimation using amino acid racemization. Indian J Dent Res 2008;19:172-4

How to cite this URL:
Kiran Kumar K. Dental age estimation using amino acid racemization. Indian J Dent Res [serial online] 2008 [cited 2020 Sep 24];19:172-4. Available from: http://www.ijdr.in/text.asp?2008/19/2/172/40478
The first steps in the identification of a cadaver are determination of sex, age, race, and stature. Estimation of age is important in identification of human remains as it enables us to construct a mortality profile of the deceased, be it in ancient archaeological populations or in contemporary forensic cases. The assignment of age is, therefore, a crucial process and estimates should be as accurate as possible. [1] The estimation of chronological age has been performed by various methods in forensic sciences. If the body is in a good condition, the apparent age can perhaps be assessed visually, but if the remains are degraded in any way, traditional morphological or histological ageing techniques on bone or dental elements need to be used. Unfortunately, traditional methods for ageing adult cadavers are highly subjective. [2] Amino acid racemization based on age-dependent, nonenzymatic changes of L-form amino acid to D-form amino acids is considered to be one of the most reliable and accurate methods. [3]

There are several age-related changes that occur in proteins, such as oxidation, isomerization, and racemization. 'Among these changes, racemization is the first-order chemical reaction from L-form to D-form and correlates highly with protein age.' [3] In the living body, newly synthesized proteins are normally composed of L-form amino acids, although there are some exceptional peptides that are biologically synthesized using D-form amino acids. In general, L-form amino acids within proteins change to D-form by an automatic chemical reaction (i.e., spontaneous conversion) whose rates are influenced by various factors such as temperature, humidity, and pH. Therefore, for estimation of chronological age, organs with low metabolic rates, such as the teeth, bone, cartilage, brain cells, and crystalline lens, are better suited for age estimation than those with high metabolic rates. [3]

Helfman and Bada in 1976 focused on aspartic acid racemization as these exhibited the most rapid racemization among the amino acids. [4] They correlated the ratio of L-amino acid and D-amino acids (D/L ratio) in dentine to age and obtained excellent results (r = 0.979). Other studies that explored aspartic acid racemization confirmed these results on tooth dentine. [5] In an attempt to further validate these findings and obtain more detailed information, Ogino and Ogino examined racemization on unerupted and supernumerary teeth, [6] while Ohtani and Yamamoto studied racemization of enamel in addition to dentine. [7]


   Choice of Organs Top


At present, based on accuracy, simplicity, and the time required, teeth are the best organ for the estimating age. However, depending on the circumstances, one can choose from other organs such as bone, cartilage, or the eye lens for estimating the D/L ratio. Among dental tissues, enamel, dentine, and cementum may be used for racemization; however, dentine is considered ideal for the purpose. [8]


   Factors Responsible for Racemization Top


Two factors which affect racemization are sample strategy and sample handling.

Sample strategy

The type of teeth chosen for the analysis may influence the results of estimation of age at death since dentinal age is not equivalent to chronological age. [1] Moreover, if the tooth has been in the oral cavity for a long duration, as in the elderly, the influence of ambient temperature is stronger and that of the period of tooth formation weaker. The mean level of D-aspartic acid in each type of tooth decreases in the following order: first molar > second molar > second premolar > first premolar > canine > central incisor > lateral incisor. [9] The coronal portions of supernumerary and impacted teeth have also been assessed for racemization and it was observed that there were some variations in the D/L ratio. [6] Burned remains will show high D/L ratio because the temperature in the fire will enhance the conversion rate of L-amino acids to the D-form. [10] As dentine forms from the crown toward the root apex, the D/L ratio should be higher in the crown and decrease toward the root apex. [11]

Sample handling

Sample handling is vital since fixation influences racemization. Ohtani et al . worked with fixatives such as ethanol and formalin and have recommended these fixatives as the standard for dental age estimation. [12] However, Waite et al . used tooth specimens which were fixed in formaldehyde. [1] This is known to cross-link proteins, particularly at amino, amide, and guanidine side chains and has the potential to fix soluble proteins to the insoluble (collagen-rich) matrix. Therefore, formaldehyde fixation influences racemization in human dentine. Although these authors were unable to obtain reproducible results from the storage of dentine in formaldehyde, they suggest that the racemization induced during acid hydrolysis is altered in the acid-soluble extract of formaldehyde-stored dentine and therefore the use of formalin fixatives is not advisable. [1] There are some enzymes which influence racemization, such as staphylococcal hydrolyse-V8. This enzyme splits only peptide bonds shared by L-glutamic or L-aspartic acid, which leads to more amounts of D-aspartic acid residues at advanced ages. [13]

Bleach treatment

In this procedure, adherent soft tissue is removed from the teeth, using sodium hypochlorite. [14] Hypochlorite penetrates the dentine, causing oxidation selectively and prolonged exposure to it may alter the D/L ratio. [1]

Washing solution

The tooth samples are washed with acetone followed by 0.2 M HCl. It is preferable to wash the samples with acetone since phosphophoryns (PPs) and low molecular weight amino acids are prone to removal by the acid wash. [7]

Pulverization

Calcified tissue is often pulverized during sample preparation prior to demineralization; pulverization increases the amount of extracted organic material that contains more soluble collagen. [15] Proteins are extracted using a chelating agent such as ethylene diamine tetra-acetic acid (EDTA) rather than mineral acids.

Demineralization

In order to isolate a fraction of the total dentine protein, demineralization is required, which is usually achieved by mineral acid (HCl) or EDTA. A concentration of 0.6 N HCl, with continuous agitation at 4C, causes much faster and simpler demineralization and can be used for dentine fragments, without the use of protease inhibitors. It is essential that low temperatures be used throughout preparation since a high temperature causes peptide bond hydrolysis. According to Carolan et al ., the disadvantage with EDTA is that it is time consuming and has no advantage over HCL. [16]

Hydrolysis

The temperature for hydrolysis ranges from 100-110C and the time periods can range from 6-20 h. However, longer-duration hydrolysis induces greater rates of racemization, whereas a shorter hydrolysis time at higher temperatures induces minimal racemization. [1]

Chromatographic separation

Both high pressure gas chromatography (HPGC) and gas chromatography (GC) have been used to separate and quantify D- aspartic and L-aspartic enantiomers in dentine. [17] GC is the method typically used in forensic settings. The amino acids are usually derived as N-trifluoroacetic acid isopropyl esters and all the amino acids can be separated and quantified on a chiral capillary column in one chromatographic run. [1] A disadvantage of GC is the sensitivity of the stationary phase to oxygen and the co-elution of a Hyp derivative with L-aspartic acid. Calibration standards for the method should be carefully chosen and those appropriate for the method used. [2]

Recommendations for standardization

  1. Individual calibration curves should be composed for each tooth type.
  2. Samples should be decontaminated.
  3. Sodium hypochlorite treatment by soaking the samples overnight in an undiluted solution of 12% w/ v chlorine is recommended.
  4. The tooth can be stored in dry air at low temperatures.
  5. Washing with dilute acid should be avoided since it leads to preextraction of acid soluble peptides.
  6. Low temperature (4C) 0.6 N HCL is used for demineralization.
  7. Hydrolysis at 100C for 6 h, using 6 M HCL.



   Conclusion Top


Aspartic acid racemization has been proposed as a means of age estimation in forensic practice for 30 years. Over the last decade, a number of laboratories around the world have developed independent protocols. While these are reasonably accurate, they have limitations in comparability, reproducibility, and evaluation. By adopting the recommendations outlined above, reliable results with total dentine aspartic acid is possible in any laboratory. The recommendations may be the first step towards standardization of the method to enable quality assurance within and between laboratories. Despite the utility of the method, the underlying chemical processes that lead to increase in racemization of aspartic acid remains unclear, without which further attempts at refining the method will be undermined.


   Acknowledgment Top


The author wishes to thank Dr. Ashith B. Acharya from the Department of Forensic Odontology at S.D.M. College of Dental Science, Dharwad, for his valuable guidance in the preparation of this review.

 
   References Top

1.Waite ER, Collins MJ, Van Duin AC. Hydroxyproline interference during the gas chromatographic analysis of D/L aspartic acid in human dentin. Int J Leg Med 1999;112:124-31.  Back to cited text no. 1    
2.Waite ER, Collins MJ, Ritz-Timme S, Schutz HW, Cattaneo C, Borrman HI. A review of the methodological aspects of aspartic acid racemisation analysis for use in forensic science. Forensic Sci Int 1997;103:113-24.  Back to cited text no. 2    
3.Ohtani S, Yamamoto T. Strategy for the estimation of chronological age using the aspartic acid racemisation method with special reference to coefficient of correlation between D/L ratios and ages. J Forensic Sci 2005;50:1020-7.  Back to cited text no. 3  [PUBMED]  
4.Helfman PM, Bada JL. Aspartic acid racemisation in dentine as a measure of ageing. Nature 1976;262:279-81.  Back to cited text no. 4  [PUBMED]  
5.Ogino T, Ogino H, Nagy B. Application of aspartic acid racemisation to forensic odontology: Post mortem designation of age at death. Forensic Sci Int 1985;29:259-67.  Back to cited text no. 5  [PUBMED]  
6.Ogino T, Ogino H. Application to forensic odontology of aspartic acid racemisation in unerupted and supernumerary teeth. J Dent Res 1988;67:1319-22.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Ohtani S, Katsuichi Y. Estimation of age from a tooth by means of racemisation of an amino acid especially aspartic acid-comparison of enamel and dentin. J Forensic Sci 1992;37:1061-7.  Back to cited text no. 7    
8.Ohtani S, Katsuichi Y. Age estimation using the racemisation of amino acid in human dentin. J Forensic Sci 1991;36:792-800.  Back to cited text no. 8    
9.Ohtani S, Ito R, 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;117:149-52.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Ohtani S, Yamada, Yamamoto I. Age estimation from racemisation rate using heated teeth. J Forensic Odonto-Stomatol 1997;15:9-12.  Back to cited text no. 10    
11.Ohtani S. Estimation of age from dentin by using the racemisation reaction of aspartic acid. Am J Forensic Med Pathol 1995;16:158-61.  Back to cited text no. 11  [PUBMED]  
12.Ohtani S, Hiroshi O, Asaka W. Estimation of age from teeth by amino acid racemisation influence of fixative. J Forensic Sci 1997;42:137-9.  Back to cited text no. 12    
13.Sajdok J, Pilin A, Pudil F, Zνdkovα J, Kαs J. A new method of age estimation based on the changes in human non-collagenous proteins from dentin. Forensic Sci Int 2006;156:245-9.  Back to cited text no. 13    
14.Carolan VA, Gardner ML, Pollard AM. Age estimation via measurement of amino acid racemisation in dental tissue. In : a sinclair, Journal of Gowlett (Edition), Proceedings of a conference on the application of scientific techniques to the study of archaeology. Archaeological Science 1995, Oxford Oxbow Monograph, Vol.64, Oxbow Books: 1997. p. 349-57.  Back to cited text no. 14    
15.Waite ER, Collins MJ, Ritz-Timme S, Schutz HW, Cattaneo C, Borrman HI. A review of the methodological aspects of aspartic acid racemisation analysis for use in forensic science. Forensic Sci Int 1997;103:113-24.  Back to cited text no. 15    
16.Carolan VA, Gardner ML, Lucy D, Pollard AM. Some considerations regarding the use of amino acid racemisation in human dentin as an indicator of age at death. J Forensic Sci 1997;42:10-6.  Back to cited text no. 16  [PUBMED]  
17.Johnson BJ, Miller GH. Archaeological applications of amino acid racemisation. Archaeometry 1997;39:265-87.  Back to cited text no. 17    

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Correspondence Address:
K Kiran Kumar
Department of Oral and Maxillofacial Pathology, SDM College of Dental Sciences and Hospital, Dharwad - 580 009, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.40478

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    Choice of Organs
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