Age Estimation by Amino Acid Racemization in Human Teeth

Abstract: When an unidentified body is found, it is essential to establish the personal identity of the body in addition to investigating the cause of death. Identification is one of the most important functions of forensic dentistry. Fingerprint, dental, and DNA analysis can be used to accurately identify a body. However, if no information is available for identification, age estimation can contribute to the resolution of a case. The authors have been using aspartic acid racemization rates in dentin (D‐aspartic acid/L ‐ aspartic acid : D/L Asp) as an index for age estimation and have obtained satisfactory results. We report five cases of age estimation using the racemization method. In all five cases, estimated ages were accurate within a range ±3 years. We conclude that the racemization method is a reliable and practical method for estimating age.     

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

Age estimation has been often performed based on the rate of aspartic acid racemization using bones. In this study, we investigated various kinds of bone and a cartilage for suitability to estimate age by racemization. Ten male cadavers aged 22 to 77 years at death were selected, and bone specimens and cartilage were taken from seven sites in each individual. The rate of racemization of aspartic acid among total amino acid contained in each specimen was analyzed by gas chromatography. The correlation coefficient between the rate of racemization and chronological age was relatively high in the sternum, skull, and femur. The rates of aspartic acid racemization were high in the costal cartilage, femur, and skull. In addition, we found that the rate of racemization was only slightly lower after the second irrigation than after the first irrigation in femur and skull bone specimens, but those of others were significantly lower. These findings showed that among the six different bones and rib cartilage, the skull and femur might be used most effectively for age estimation using total amino acid fraction.     

Age estimation of bloodstains: a preliminary report based on aspartic acid racemization rate

This study describes an innovative application of a well-established method of age determination. The conventional method of aspartic acid racemization (AAR) is based on estimation of the d-l-aspartic acid ratio in slow turnover tissues, such as tooth tissue, to reflect the age of an individual. This method has been recently applied to age estimation in forensic investigations, and is also widely used for archeological dating of fossils. We suggest that the aspartic acid racemization method could be applied to a significant, although unresolved, forensic issue: that of bloodstain dating. Standard kinetic experiments were used to describe the characteristics of the racemization reaction in bloodstains, which were then employed to estimate the age of various samples. The soluble protein fraction of a bloodstain produced a stronger correlation between elapsed time and d-aspartic acid content than total amino acid fractions. According to our preliminary results, the time lapse after the creation of a bloodstain can be determined ex vivo by measuring the extent of aspartic acid racemization. Our analysis highlights the need for further study into the preservation and composition of bloodstains to assist in further development of this pioneering application.     

Application of aspartic acid racemization to forensic odontology: post mortem designation of age at death

The extent of racemization of aspartic acid in the dentin of healthy, carious and impacted teeth was used to define the ages of individuals at the time of their death. These determinations were performed with an error of +/- 4 years. This method holds the promise of simplifying procedures in, and extending the application of forensic odontology. The current error range of this method may be reduced by analyzing teeth of the same positional types from deceased individuals of known age. Using this method, it appears that post mortem preservation of up to 10 years has a negligible effect on the values determined for the age of individuals at death.     

Estimation of age from a tooth by means of racemization of an amino acid, especially aspartic acid--comparison of enamel and dentin.

In a study of age estimation from teeth by means of measuring racemization of aspartic acid (Asp), a representative amino acid, the accuracy of data from enamel and dentin in the same tooth was compared. The correlation of D/L ratio of aspartic acid with actual age gave the following parameters: r = 0.928, sigma = +/- 5.2, k = 4.47 x 10(-4) yr-1 in enamel and r = 0.995, sigma = +/- 1.4, k = 5.75 x 10(-4) yr-1. The difference in ages between one estimated by the D/L ratio and the actual one was within +/- 3 years in dentin, while in enamel an error of from 2 to 11 years was observed. Reaction rate constants of the racemization in a dry postmortem state (15 degrees C) were calculated as k = 9.70 x 10(-8) yr-1 in enamel, and k = 1.33 x 10(-7) yr-1 in dentin. Compared to rates determined from teeth recently extracted from living subjects, the rate was higher in dentin than in enamel. These data reconfirmed that dentin is superior to enamel in making exact age estimations from teeth.     

Racemization of aspartic acid from human dentin in the estimation of chronological age

The estimation of chronological age in cadavers, human remains and in living human beings by various methods is discussed. These methods, which are based on the age dependent non-enzymatic changes of l-form amino acids to d-form amino acids, mainly aspartic acid, are among the most reliable and accurate methods to date. Most of these methods use gas chromatography (GC). In this review, results of aspartic acid racemization in dentin at different targets are discussed. In addition, pre-considerations and guidelines are given for the selection of dentin from teeth. A pilot project was run to evaluate the efficiency of high performance liquid chromatography (HPLC) coupled with fluorescence detection. New buffer conditions were found to obtain stable derivatives of aspartic acid enantiomers for the estimation of racemization.     

A new approach to amino acid racemization in enamel: testing of a less destructive sampling methodology

Aspartic acid racemization has been found to be an accurate measure of age at death for recent forensic material. This paper examines the practicality of using acid etching of the tooth surface to extract amino acids from the enamel for racemization analysis. By serial etching of the tooth and contamination of the teeth with bovine serum albumin prior to etching, the ability of etching to remove contamination was assessed. The destructiveness of the method was visualized and quantified using micro-computed tomography (micro-CT). By bleaching the teeth and by deeper etching it was possible to obtain more consistent values. While etching had little effect on the enamel at the macroscale, it did have an impact at the microscale. The quantities of enamel removed varied depending upon the tooth morphology, but were not large. Acid etching of enamel thus appears to be a promising new method for extracting proteins for amino acid racemization age estimation noninvasively.     

The extent of aspartic acid racemization in dentin: a possible method for a more accurate determination of age at death?

In the current study the objective was to find to what extent a reliable determination of age at death is made possible by establishing the degree of aspartic acid racemization in the dentin of teeth. The results of the investigation of 46 teeth are in agreement with the values found by other authors. The method presented makes a reproducible and accurate estimation of age possible. We intend to elaborate and improve this promising method for determination of age at death. The relevant points are presented here.     

A review of the methodological aspects of aspartic acid racemization analysis for use in forensic science.

Accurate age determination of adult cadavers and human remains is a key requirement in forensic practice. The current morphological methods lack accuracy and precision, require specialist training and are costly. The use of aspartic acid racemization (AAR) in human dentine provides a simple, cost-effective solution and the method can achieve accuracies of +/- 3 years at best. Currently, there are differences in AAR methodology between laboratories which produce different results on the rate of racemization in teeth. These inconsistencies must be resolved if the technique is to be successfully applied to age determinations in forensic cases. This paper reviews the differences in protocol which have been used, discusses how each method will affect the results obtained from AAR analysis and gives recommendations for optimization of the methological protocol as a first step towards international standardization.     

The application of amino acid racemization in the acid soluble fraction of enamel to the estimation of the age of human teeth

Estimation of age-at-death for skeletonised forensic remains is one of the most significant problems in forensic anthropology. 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 racemization in dentine. However, this method cannot be used on remains where the post-mortem interval is greater than 20 years. An alternative approach is to measure amino acid racemization in dental enamel, which is believed to be more resistant to change post-mortem. The extent of amino acid racemization in the acid soluble fraction of the enamel proteins was determined for modern known age teeth. A strong correlation was observed between the age of the tooth and the extent of racemization. No systematic bias in the direction of age estimation errors was detected. For the majority of teeth analyzed, the presence of dental caries did not affect the results obtained. In a minority of cases, carious teeth showed a higher level of racemization than would be expected given the age of the individual. These results indicate that amino acid racemization in enamel has the potential to be used in age estimation of skeletal remains.     

Comparison of age estimated from degree of racemization of aspartic acid, glutamic acid and alanine in the femur

Aspartic acid (Asp) is generally used for estimation of age by measuring the degree of racemization. For other amino acids, however, there are few reports regarding the usefulness of the degree of racemization for the estimation of age. Accordingly, in this study using the femur (obtained from 21 cadavers) as the specimen, we measured the degree of racemization of glutamic acid (Glu) and alanine (Ala) along with Asp in the total amino acid (TAA) fraction as well as in acid-insoluble collagen-rich (IC) and acid-soluble peptide (SP) sub-fractions. We compared the degrees of racemization of each amino acid and the accuracy of the ages estimated from them. The degree of racemization and the reaction rate of racemization were ranked in the order of Asp > Glu > Ala in the TAA and IC fractions, but Asp > Ala > Glu in the SP fraction. It is noteworthy that the degrees of racemization differed between the three amino acids depending on the fraction tested. The correlation coefficient (r) between the degree of racemization and the chronological age was higher in the SP than in the TAA or IC fraction. Among three amino acids, Asp showed the highest correlation coefficient as predicted. The present study confirmed that Asp from the SP fraction is the best indicator for age estimation using racemization rates.     

Age estimation using the racemization of amino acid in human dentin

This study was made to improve the validity of age estimation from teeth using amino acid racemization. The correlation between actual age and the D/L ratio of aspartic acid was investigated by analyzing not only the total amino acid but also its fractionated substances, insoluble collagen and soluble peptide. The coefficient values of correlation between the D/L ratio and actual age in adult lower central incisors were 0.996 (sigma = +/- 1.0 year) for the total amino acid, 0.988 (sigma = +/- 1.8 years) for insoluble collagen, and 0.997 (sigma = +/- 0.9 years) for soluble peptide. The corresponding figures in adult first premolars were 0.991 (sigma = +/- 1.6 years), 0.988 (sigma = +/- 1.9 years), and 0.994 (sigma = +/- 1.4 years), respectively. The reactive velocity of aspartic acid racemization was highest for soluble peptide in both the lower central incisors and first premolars and approximately three times as rapid as that for total amino acid. As a result, age estimated from the analysis of soluble peptide was most accurate. The velocity for insoluble collagen was slightly lower than that for total amino acid. Age estimation was attempted from the teeth of an unknown body. These results suggest that the analyses, not only of total amino acid in dentin but also of its fractionated and extracted substances, can lead to higher reliability in age estimation. Soluble peptide, in particular, has been found to be most effective.     

Age Estimation by Racemization Method in Teeth: Application of Aspartic Acid,Glutamate, and Alanine

Abstract: Here, we report on an experimental approach of simultaneous determination of various amino acids racemization (AAR) rates in teeth. We evaluated the measurements of aspartic acid (Asp), glutamate (Glu), and alanine (Ala) isolated from dentin. Asx D/L rates from total amino acid fraction, generally used for age estimation, showed high correlation (r = 0.98) with age. As Glx and Ala showed very slow racemization kinetics in TA, we performed further analysis of the acid‐soluble protein (SP) fraction. The results supported improved correlation between age and D/L rates for Glu (r = 0.84) and Ala (r = 0.85), as well as for Asp (r = 0.98). By providing further elucidation on dentin protein racemization, the technique offers a considerable opportunity to involve other amino acids in age estimation studies. As the process does not require additional separation steps, the method can be easily adapted to existing protocols.     

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

The estimation of chronological age has been performed by various methods in forensic science. Among these, racemization methods, which are based on the age-dependent non-enzymatic changes of L-form amino acids to D-form mainly using aspartic acid, are one of the most reliable and accurate methods to date. Separation of enantiomers is generally performed by gas chromatography or high performance liquid chromatography. Various tissues with low metabolic rates have been applied for this purpose. In addition, single proteins purified from these target tissues are also applicable. In this brief review we describe this method in detail, noting points of caution, as well as the advantages and disadvantages of the different target tissues. In addition, special attention is given to the correlation rates obtained between chronological age and enantiomer ratios. Currently, based on accuracy of estimated age, simplicity of the method, time required, and reproducibility, tooth dentin is considered one of the best target tissues. Alternatively, analysis of osteocalcin and elastin have also provided accurate and reproducible results.     

Comparison of Age Estimation in Japanese and Scandinavian Teeth Using Amino Acid Racemization*

Abstract: Racemization method is currently considered to be one of the most accurate methods for age estimation. There are many factors that affect racemization reaction velocities. We investigated ethnic differences in the racemization reaction velocities between Japanese and Scandinavian teeth. After a heating experiment, the Arrhenius equation was used to obtain a rate constant (k). Both groups presented almost identical velocities, indicating that there was no difference in the tooth racemization reaction between the two groups. However, because 14 of the 18 Scandinavian teeth had multiple roots, it is possible that accurate racemization rates of the teeth themselves were not reflected in the results. These findings reconfirm that the type of tooth selected is extremely important when evaluating age by racemization.     

An application of D- and L-aspartic acid mixtures as standard specimens for the chronological age estimation

Instead of using the control teeth, we tried to prepare standard specimens to derive the equations for calculation of the age (analytical curves) with respect to each kind of tooth of various ages. To prepare standard specimens, we determined the racemization ratio of the teeth of known age that had actually been used for the appraisal of chronological age (total control teeth). Then we mixed commercially available L-Asp and D-Asp in the same D/L ratio as the measured one in the total control teeth. As a result, we were able to obtain the racemization rate equations from the age-specific standard specimens of central and lateral incisors. These equations were closely similar to those derived from actual teeth of known age. Since the racemization rate equations obtained from the standard specimens were satisfactorily reproducible, we assumed that these equations could be used in place of those obtained from the control teeth. Actually, in the age estimation of unidentified corpses from teeth, the use of standard specimens enabled us to estimate the age almost as precisely as estimated using the control teeth. Thus, the present study has demonstrated that in the estimation of chronological age the control teeth can be substituted by the standard specimens. This shows the possibility of using the standard specimens also in other laboratories where the racemization ratio can be measured with sufficient reproducibility.     

气相色谱法测定牙本质中天冬氨酸消旋化程度推断年龄的研究

本文应用手性毛细管柱气相色氨谱法，以酸性异丙醇、三氯乙酸酐为衍生化试剂，对不同年龄人的27颗第一磨牙牙本质中D型与L型天冬氟酸的浓度进行了测定，并计算了二者的比率，建立了消旋化程度与年龄的回归方程，r＝0．9568，SD＝4．5岁，本研究为用牙齿推断人的年龄提供了一个比较客观、准确的新手段。     

Age estimation from aspartic acid racemization of root dentin by internal standard method

In this study, we investigated the application of the internal standard method to determine age from aspartic acid (Asp) racemization. D-Methionine (D-Met) and D-norleucine (D-Nleu) were tested as internal standards for the purpose of validating the derivatization and gas chromatographic measurements. Using a set of standard amino acids plus the internal standards in constant volume, calibration plots with reasonable linearity (R > 0.98) were constructed. Based on the analysis of sample chromatograms, D-Met appeared to meet the criteria for internal standards, hence it was selected for use in D- and L-Asp quantification. The correlation between dentin age and D-/L-Asp ratios from the peak areas as well as from the absolute concentrations was investigated. Correlation coefficients were calculated as 0.98 and 0.90, respectively. The slight decrease in accuracy was attributed to the conversion of D-Asp/D-Met ratios to concentrations employing the calibration curves figured from pure Asp. Because the application of the internal standard method produced reproducible and precise measurements, the employment of internal standards in age estimation based on Asp racemization appears to provide quality assurance by avoiding possible errors arising from sample preparation.     

The Effects of Racemization Rate for Age Estimation of Pink Teeth

Pink teeth is thought to result from the seepage of hemoglobin caused by dental pulp decomposition. We investigated whether racemization can be applied for age estimation in cases of pink teeth where the whole tooth is used. The pink teeth used were three cases and the normal teeth for control were five mandibular canines of known age. Age of the pink teeth was calculated on the basis of regression formula obtained from the five control teeth. Only a slight error was noted between the actual and estimated ages of the pink teeth (R² = 0.980, r = 0.990): Cases 1–3 actually aged 23, 53, and 59 years were estimated to be 26, 52, and 60 years. Based on our results of testing pink teeth of known age, we suggest that racemization techniques allow for the age estimation of pink teeth using the same methods for normally colored teeth.     

Pediatric toxicologic deaths: a 10-year retrospective study

A 10-year retrospective study of pediatric toxicologic deaths was performed at the Medical University of South Carolina (Charleston, SC) from January 1989 to December 1998. During this time, 709 pediatric forensic autopsies were performed on children younger than 18 years of age. Eleven deaths were determined to be secondary to toxic exposures (excluding carbon monoxide poisonings secondary to fires). The remaining deaths were reviewed for the presence of alcohol or illicit drugs. The 11 toxicologic deaths were analyzed for age, sex, race, type of toxic exposure, cause and manner of death, location of incident, witness, and, in the younger age group, the primary caregiver at the time of exposure. The deaths had a bimodal age distribution (6 deaths in victims ages 15 to 17 and 5 deaths in victims ages 4 or younger), involving a wide range of toxins. The teenage group was composed of five males and one female, all white. The preschool group had three females and three males, all black. The manner of death ranged from accidental to suicidal to homicidal. In addition, in eight neonatal and fetal deaths, the victims tested positive for maternal cocaine use, and five of these victims tested positive for cocaine or benzoylecgonine. However, the cause of death was not stated to be cocaine in any of these neonatal and fetal cases.