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.     

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 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.     

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.     

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 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.     

Estimation of Chronological Age from Postmortem Tissues Based on Amino Acid Racemization

Skin and cartilage tissue specimens from 32 male and 13 female corpses aged between 17 and 50 years were collected within 24 h after the death. Each specimen was analyzed for the composition of dextro (D) and levo (L) forms of aspartate, glutamate, and alanine. Linear regression models were constructed using ln [(1 + D/L)/(1 ? D/L] equation to define the relationship between the extent of racemization and the chronological age. Aspartate D/L rates from cartilage showed high correlation (r = 0.779, p < 0.001, n = 45). Aspartate D/L rates from skin showed very low correlation (r = 0.356, p < 0.002, n = 44). The multilinear regression model of both aspartate D/L rates of cartilage and skin tissues in 44 cases yielded a coefficient of r = 0.828 (p < 0.001). In conclusion, only racemization rate of Aspartate both in the skin and the cartilage tissues correlated with the chronological age. Our results may imply that the age can be estimated more precisely if two different tissue specimens are obtained from one corpse.     

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 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.     

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.     

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 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.     

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.     

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 use of the D-, L- aspartic ratio in decalcified collagen from human dentin as an estimator of human age

Among the methods dealing with the age estimation, the evaluation of the ratio of the D-, L- form of the aspartic acid in tissues with a low metabolic turnover is considered to be the most precise. We introduced demineralization of the dentin with 0.5 M EDTA adjusted to pH = 7.4. The advantage of such a procedure is that after demineralization we obtained pure insoluble protein (collagen) and soluble noncollagenous proteins in one step. In this study we analyzed insoluble collagen. The amino acids obtained after the hydrolysis were derivatized into TFA isopropyl esters and analyzed by gas chromatography on Chirasil L-Val capillary column. We analyzed human dentin from the lower canines. The correlation coefficient was 0.93 for our set of 71 persons. The result concurred with those of other scientists.     

Molecular Autopsy of Desmosomal Protein Plakophilin‐2 in Sudden Unexplained Nocturnal Death Syndrome

Plakophilin‐2 (PKP2) variants could produce a phenotype of Brugada syndrome (BrS), which seems to be most likely the same allelic disorder as some sudden unexplained nocturnal death syndrome (SUNDS). All coding regions of PKP2 gene in 119 SUNDS victims were genetically screened using PCR and direct Sanger sequencing methods. Three novel mutations (p.Ala159Thr, p.Val200Val, and p.Gly265Glu), one novel rare polymorphism (p.Thr723Thr), and eight reported polymorphisms were identified. A compound mutation (p.Ala159Thr and p.Gly265Glu) and a rare polymorphism (p.Thr723Thr) were found in one SUNDS case with absence of the cardiomyopathic features. The detected compound mutation identified in this first investigation of PKP2 genetic phenotype in SUNDS is regarded as the plausible genetic cause of this SUNDS case. The rare incidence of PKP2 mutation in SUNDS (1%) supports the previous viewpoint that SUNDS is most likely an allelic disorder as BrS.     

HPLC测定牙本质中天冬氨酸消旋化程度及在年龄推断上的应用

用邻苯二甲醛-N-乙酸-L一半脱氨酸将牙本质中氨基酸对映体衍生成非对映体，其产物同HPLC分离，荧光检测器检测，20颗人类第一磨牙本质中天冬氨酸的D／L值，其与实际年龄的相关系数为0．9889，误差在±1岁以内者占40％，最大误差为±4岁。     

The Reliability of Chronological Age Determination by Means of Mandibular Third Molar Development in Subjects in Croatia*

Abstract: The aim of this study was to determine the development of the mandibular third molar and its relation to chronological age. The evaluated material consisted of 979 orthopantomograms of patients of Croatian Caucasian origin aged between 5.7 and 14.6 years. Third molar developmental stages were evaluated according to the stages proposed by Nolla. The frequencies of different stages of mineralization and the mean value of the mineralization of the mandibular third molars with regard to age, gender, and size of the mandible were determined and the coefficient of correlation determined between the age of the subject and the stage of development of the mandibular molars. Statistically significant correlation was determined between mineralization of the mandibular third molar and chronological age of the subjects (boys, mandibular left third molar r = 0.779, right third molar r = 0.793; girls, mandibular left third molar r = 0.746, right third molar r = 0.725). It can be concluded that the accuracy of age estimation based on Nolla’s method is applicable for Croatian children.     

Differentiation of Morphologically Similar Human Head Hairs from Two Demographically Similar Individuals Using Amino Acid Ratios,

Human hair is frequently encountered as forensic evidence and can contribute valuable information to investigators. Conventional forensic hair analyses include microscopic hair comparison (MHC) and DNA analysis. However, MHC is not supported by statistics and DNA analysis cannot always be performed. Recent studies have demonstrated that evaluation of differences in the hair proteins may offer an alternate method to these analyses. In this study, an evaluation of the amino acids present in hair was investigated as an approach to differentiate morphologically indistinguishable hair samples from two demographically similar individuals. Proteins in the hair were digested using hydrochloric acid, and the resulting amino acids were derivatized with N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) for analysis using gas chromatography-mass spectrometry (GC-MS). Eight derivatized amino acids were detected and quantified relative to an internal standard, L-norvaline, and used to construct twenty-eight amino acid ratios. Hair samples were collected from four areas of the head on various days over the course of one month, and no significant differences in amino acid ratios (p-value > 0.05) were observed among the areas of the head, and the ratios were consistent over the time period of this study. Additionally, fifteen of these amino acid ratios were found to be significantly different between the two individuals when compared using a two-sample t-test (p-value ≤ 0.05). These data indicate that amino acid analysis was able to differentiate two morphologically similar hair samples from different individuals and demonstrates the applicability of this method to distinguish similar hair samples when DNA analysis cannot be performed.