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.     

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.     

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.     

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.     

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.     

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 in children by measurement of open apices in tooth roots: Study of a Mexican sample

The aim of this cross-sectional study was to test the accuracy of Cameriere's European formula for age assessment in a large sample of Mexican children. The accuracy of dental age estimation was defined as how closely real age, measured as the difference between chronological age (CA) and dental age (DA), could be predicted. Digitalized orthopantomographs of 502 Mexican children (254 girls and 248 boys), aged between 5 and 15 years, were analyzed. The seven left permanent mandibular teeth were evaluated using Cameriere's method. Intra- and inter-observer variability for this technique was tested on a small random sample. Dental age was estimated for each individual and compared with known chronological age. Accuracy was measured as the difference between known chronological age and dental age and tested for significance with the mean prediction error (ME). The standard deviation and 95% confidence interval of the mean difference were also calculated. ME was 0.63 years for girls and 0.52 years for boys. ME was found to be slightly overestimated by 0.10 years for girls, but was correctly estimated for boys with an accuracy of 0.00. In conclusion, this method is very useful and may be recommended for practical application both in clinical dentistry and forensic procedures on the Mexican population.     

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.     

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.     

Approaches to chronological age assessment based on dental calcification

The aim of the present study was to find an accurate estimation of chronological age using a small number of selected teeth. For this purpose, the method devised by Nolla [C. Nolla, The development of the permanent teeth, J. Dent. Child. 27 (1960) 254-266.] was used: the development of each of the teeth was determined according to this method on 374 radiographs, 195 of boys (mean age 8.59) and 179 of girls (mean age 8.75). The 28 variables representing the calcification stages were analyzed using cluster analysis followed by multivariate analysis (multiple linear regression model). Patient age was considered to be a dependent variable. Our study showed that antimere teeth are the most homogeneous as regards stages of development. The prediction was more accurate for boys and girls below 10 years of age, using teeth 21, 43 and 46 from boys and teeth 21, 46 and 47 from girls. These teeth accounted for 80% total variance of chronological age for dental calcification. Standard error was +/-1.4 years for boys and +/-1.2 years for girls. When the age of the children remained completely unknown, the best estimates were provided by teeth 43, 47, 46 and 44 from boys and teeth 44, 47 and 43 from girls.     

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.     

Age calculation using X-ray microfocus computed tomographical scanning of teeth: a pilot study

To correlate dental age with an individual's chronological age based on the calculated volume ratio of pulp versus tooth volume measured, an X-ray microfocus computed tomography unit (microCT) with 25 microm spatial resolution was used to non-destructively scan 43 extracted single root teeth of 25 individuals with well-known chronological age. Custom-made analysis software was used by two examiners to obtain numerical values for pulpal and tooth volume. The ratio of both was calculated and statistically processed. No significant intra- or inter-examiner differences were found. In fact, a very strong concordance correlation coefficient was found. Linear regression analysis showed a coefficient of determination (r) of 0.31 which suggests that there is a rather weak correlation between the volume ratio of pulp versus tooth and biological age. Although rather time consuming, this technique shows promising results for dental age estimation in a non-destructive manner using X-ray microfocus computed tomography.     

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.     

The accuracy of three methods of age estimation using radiographic measurements of developing teeth

The accuracy of age estimation using three quantitative methods of developing permanent teeth was investigated. These were M?rnstad et al. [Scand. J. Dent. Res. 102 (1994) 137], Liversidge and Molleson [J. For. Sci. 44 (1999) 917] and Carels et al. [J. Biol. Bucc. 19 (1991) 297]. The sample consisted of 145 white Caucasian children (75 girls, 70 boys) aged between 8 and 13 years. Tooth length and apex width of mandibular canine, premolars and first and second molars were measured from orthopantomographs using a digitiser. These data were substituted into equations from the three methods and estimated age was calculated and compared to chronological age. Age was under-estimated in boys and girls using all the three methods; the mean difference between chronological and estimated ages for method I was -0.83 (standard deviation +/-0.96) years for boys and -0.67 (+/-0.76) years for girls; method II -0.79 (+/-0.93) and -0.63 (+/-0.92); method III -1.03 (+/-1.48) and -1.35 (+/-1.11) for boys and girls, respectively. Further analysis of age cohorts, found the most accurate method to be method I for the age group 8.00-8.99 years where age could be predicted to 0.14+/-0.44 years (boys) and 0.10+/-0.32 years (girls). Accuracy was greater for younger children compared to older children and this decreased with age.     

Dental age estimation through volume matching of teeth imaged by cone-beam CT

A custom-made voxel counting software for calculating the ratio between pulp canal versus tooth volume based on cone-beam CT tooth images was developed and evaluated. The aim of this study was to attempt establishing a correlation between the chronological age of a certain individual and the pulp/tooth volume ratio of one of the teeth. Twenty-eight single rooted teeth of 19 individuals with well-known chronological age were scanned by the cone-beam CT (3D Accuitomo, J. Morita, Kyoto, Japan). Next the images were analyzed using the custom-made software. Linear regression analysis was performed. The results of the analysis showed a moderate correlation between the pulp/tooth volume ratio and biological age with a coefficient of determination (R2) of 0.29. Although the present work was limited to a pilot study, the developed technique showed promising results for dental age estimation in a non-invasive manner using cone-beam CT images in living individuals.     

Age Estimation of Infants Through Metric Analysis of Developing Anterior Deciduous Teeth

This study provides regression equations for estimation of age of infants from the dimensions of their developing deciduous teeth. The sample comprises 97 individuals of known sex and age (62 boys, 35 girls), aged between 2 days and 1,081 days. The age‐estimation equations were obtained for the sexes combined, as well as for each sex separately, thus including “sex” as an independent variable. The values of the correlations and determination coefficients obtained for each regression equation indicate good fits for most of the equations obtained. The “sex” factor was statistically significant when included as an independent variable in seven of the regression equations. However, the “sex” factor provided an advantage for age estimation in only three of the equations, compared to those that did not include “sex” as a factor. These data suggest that the ages of infants can be accurately estimated from measurements of their developing deciduous teeth.     

Relationship Between Pulp Tooth Area Ratio and Chronological Age Using Cone‐beam Computed Tomography Images

This study investigated the relationship between chronological age and pulp tooth area ratio (PTR) in maxillary central teeth using cone‐beam computed tomography (CBCT) images in the Turkish population. The sample consisted of CBCT images of 120 male and female subjects between 14 and 75 years old. The PTR of the maxillary central teeth was calculated using inVivo 5 software (Anatomage, San Jose, CA). Data were analyzed with independent t‐test and Pearson's rank correlation test. There was a significant negative correlation between chronological age and PTR (r = ?0.615). The results showed that the PTR of maxillary central teeth is a reliable method for age estimation in the Turkish population.     

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.     

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.     

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。