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 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.     

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.     

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.     

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.     

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。     

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.     

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.     

Developing permanent tooth length as an estimate of age.

Developing teeth are widely used to predict age in archaeology and forensic science. Regression equations of tooth length for age is a direct method, however, data for permanent teeth is incomplete. The aims of this study were: (a) to calculate regression equations predicting age from tooth length of all permanent teeth from birth to maturity, and (b) to evaluate the difference between radiographic and actual tooth length. The sample studied (N = 76, age range 0 to 19 years) was the Spitalfields juveniles of recorded age-at-death. Tooth length was measured from incisal tip to developing edge of crown or root of 354 dissected teeth. Data for upper and lower teeth were combined except for the lateral incisor. The least squares regression method was used to analyze the data for each tooth type; age being regressed against tooth length for prediction. For most tooth types, growth followed an S-shaped (polynomial) curve with initial fast growth and a further growth spurt around the time of mid root formation. No difference was found between radiographic and true tooth length. These regression equations provide an easy method of predicting age from any developing permanent tooth by measuring tooth length from isolated teeth or from unmagnified, undistorted radiographs.     

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 at death determinations for autopsied remains based on aspartic acid racemization in tooth dentin: importance of postmortem conditions

The extent of aspartic acid racemization in human dentin can be used to assess chronological age of the individual. This novel chemical method of aging human remains has potential applications in forensic sciences. Six autopsy cases have been analyzed for D/L aspartic acid contents, and their ages at death calculated. Independent age information came from known identity, tooth maturation, or tooth wear estimations. The six cases represented a range of postmortem fates: recent demise, burial, and ground surface exposure. Five samples, four recently deceased and one a burial, showed racemization ages in close agreement with other age data. One specimen, subjected to 51 days of open air exposure, exhibited a vastly inflated racemization age. Postmortem conditions are crucial to the accuracy of racemization age at death measurements.     

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.     

Age estimation by occlusal tooth wear

The purpose of the present investigation was to test the accuracy of a new scoring system in recording tooth wear for age estimation. The material consisted of dental stone casts of 383 volunteers who had sound premolars and molars with normal occlusion. The degree of occlusal wear for all premolar and molar teeth was scored with the new system. The high intra- and inter-examiner concordances showed that the new score system was very reliable. The degree of tooth wear showed a significant positive correlation with age in each and every examined tooth of both males and females. Tooth wear scores of males were higher than those of females. Calculating tables for age estimation were designed and the accuracy of age estimation was obtained with the General Linear Models procedures. Our system could provide estimation of an individual's age within +/-3 years in 42.4% of males and 49.4% of females, within +/-5 years in 61.8% of males and 63.3% of females. When the subjects were divided into two age groups and data were re-treated, the accuracy of age estimation was increased. Collectively, it was shown that our new system for scoring tooth wear is a reliable and accurate method for age estimation.     

Accuracy of developing tooth length as an estimate of age in human skeletal remains: the deciduous dentition

Dental age assessments are widely used to estimate age of immature skeletal remains. Most methods have relied on fractional stages of tooth emergence and formation, particularly of the permanent dentition, for predicting the age of infants and very young children. In this study, the accuracy of regression equations of developing deciduous tooth length for age estimation (Liversidge et al.) is tested on a sample of 30 Portuguese subadult skeletons of known age at death. Overall the method shows high accuracy and the average difference between estimated and chronological age is between 0.20 and -0.14 years when using single teeth, and 0.06 years, when using all available teeth. However, there is a tendency for the deciduous molars to provide overestimates of chronological age. Results show that age estimates can be obtained within +/-0.10 years with a 95% confidence interval when several teeth are used. Overall between-tooth agreement in age estimates decreases with increasing age but there is less variability of estimates with more teeth contributing to overall mean age. One seemingly limitation of this method may be the fact that it was developed by combining the maxillary and mandibular teeth. The other is related to the accuracy with which radiographic tooth length can be used as a valid surrogate for actual tooth length. Nevertheless, the advantages of this metric method surpass the limitations of chronologies based on stages of dental development.     

中国汉族6～18周岁人群下颌恒牙钙化的性别差异研究

目的通过对中国汉族男女6～18周岁人群全口曲面断层影像上所显示的下颌左侧牙齿钙化程度进行比较研究,探究牙齿生长发育的性别差异,为建立用牙齿推断中国汉族儿童及青少年年龄的线性回归模型提供理论依据。方法通过对13491张(男6568张,女6923张)中国汉族6～18周岁儿童及青少年全口曲面断层影像的牙齿钙化规律进行研究,建立牙齿钙化的分级标准,并对下颌恒牙进行分级评分;数据用SPSS 13.0软件处理。结果非参数检验结果显示大部分年龄组下颌左侧8颗恒牙钙化分级值性别间的差异有统计学意义(P0.05);在8颗恒牙发育程度同等条件下,女性年龄比男性小0.303岁。结论中国汉族6～18周岁人群牙齿发育存在着性别差异;在利用牙齿钙化程度建立推断中国汉族儿童及青少年年龄的方程模型时,应分性别建模。     

Age estimation of Korean adults by occlusal tooth wear

The aim of the present study was to evaluate the validity of the modified Kim's scoring system as a practical method of recording occlusal tooth wear and estimating age. For this purpose, 1092 pairs of maxillary and mandibular full-arch casts were randomly selected, scored, and analyzed. The results showed that the modified Kim's scoring system had excellent reliability, and that occlusal tooth wear had a positive correlation with age. Tooth wear scores of all teeth except the two lower central incisors were higher in males than in females. Calculating tables for age estimation were designed by multiple linear regression analysis. Estimated ages were within +/-5 years of actual ages in 63.5% of male subjects, and 64.0% of female subjects. The accuracy of age estimation was increased when the subjects were divided into two age groups and data were re-analyzed. Collectively, it was shown that the modified Kim's scoring system is a reliable and accurate method for age estimation, and that the data from these 1092 individuals can be used as a standard for age estimation of Korean adults.     

Age estimation by pulp/tooth ratio in lower premolars by orthopantomography

Accurate age estimation has always been a problem for forensic scientists, and apposition of secondary dentine is often used as an indicator of age. Since 2004, in order to examine patterns of secondary dentine apposition, Cameriere et al. have been extensively studying the pulp/tooth area ratio of the canines by panoramic and peri-apical X-ray images. The main aim of this paper is to examine the relationship between age and age-related changes in the pulp/tooth area ratio in monoradicular teeth, with the exception of canines, by orthopantomography. A total of 606 orthopantomograms of Spanish white Caucasian patients (289 women and 317 men), aged between 18 and 75 years and coming from Bilbao and Granada (Spain), was analysed. Regression analysis of age of monoradicular teeth indicated that the lower premolars were the most closely correlated with age. An ANCOVA did not show significant differences between men and women. Multiple regression analysis, with age as dependent variable and pulp/tooth area ratio as predictor, yielded several formulae. R(2) ranged from 0.69 to 0.75 for a single lower premolar tooth and from 0.79 to 0.86 for multiple lower premolar teeth. Depending on the available number of premolar teeth, the mean of the absolute values of residual standard error, at 95% confidence interval, ranged between 4.34 and 6.02 years, showing that the pulp/tooth area ratio is a useful variable for assessing age with reasonable accuracy.     

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 in small children: reference values based on counts of deciduous teeth in Finns

The eruption of teeth in the mouth is suitable for age estimations during the period when teeth are actively emerging, in the deciduous dentition phase approximately from the age of 6 months to 2.5 years. Estimations of age can be performed simply by counting the number of teeth in the mouth. Reliability of the estimates depends on the reference data available and each population group should preferably have its own standards. In the present study timing of eruption of successive deciduous teeth was studied longitudinally in 129 Finns. The dates of clinical eruption of deciduous teeth were recorded by mothers and checked by dentists. In 40 of the 129 children emergence ages of at the most the four last teeth were based only on semiannual registrations performed by dentists. The main purpose was to provide normal timetables of tooth eruption in small children in forms that are practical in estimations of dental age. No sexual dimorphism existed in the timing of clinical eruption of successive deciduous teeth. The mean age corresponding to the presence of one tooth in the mouth was 7.1 months (S.D.=1.78) and that corresponding to tooth count 19 was 27.8 months (S.D.=3.99). If the chronological age is known, the presented distributions and means with variations make it possible to estimate the degree of advancement or delay in a child’s dental development. If the age of the child is not known, the mean and median ages can be used for estimations of chronological age. However, estimations of age should not be based only on tooth counts because of marked variation also within this homogeneous group.     

Age estimation in small children: reference values based on counts of deciduous teeth in Finns

The eruption of teeth in the mouth is suitable for age estimations during the period when teeth are actively emerging, in the deciduous dentition phase approximately from the age of 6 months to 2.5 years. Estimations of age can be performed simply by counting the number of teeth in the mouth. Reliability of the estimates depends on the reference data available and each population group should preferably have its own standards. In the present study timing of eruption of successive deciduous teeth was studied longitudinally in 129 Finns. The dates of clinical eruption of deciduous teeth were recorded by mothers and checked by dentists. In 40 of the 129 children emergence ages of at the most the four last teeth were based only on semiannual registrations performed by dentists. The main purpose was to provide normal timetables of tooth eruption in small children in forms that are practical in estimations of dental age. No sexual dimorphism existed in the timing of clinical eruption of successive deciduous teeth. The mean age corresponding to the presence of one tooth in the mouth was 7.1 months (S.D.=1.78) and that corresponding to tooth count 19 was 27.8 months (S.D.=3.99). If the chronological age is known, the presented distributions and means with variations make it possible to estimate the degree of advancement or delay in a child's dental development. If the age of the child is not known, the mean and median ages can be used for estimations of chronological age. However, estimations of age should not be based only on tooth counts because of marked variation also within this homogeneous group.