Postmortem estimation of age at death based on aspartic acid racemization in dentin: Its applicability for root dentin

Summary The extent of aspartic acid racemization in total dentin and in dentin protein fractions from theroots of third molars was determined. In several cases coronal dentin was also investigated. The results of other authors, according to which the racemization of aspartic acid in root dentin apparently proceeds differently than in coronal dentin, could be confirmed. Consequently, the data published so far on age determination based on the extent of aspartic acid racemization in coronal dentin and the entire dentin of longitudinal sections cannot be applied to root dentin. In total root dentin and the acid soluble protein of root dentin, a close relationship was observed between the extent of aspartic acid racemization and age. Accordingly, estimation of age at death based on aspartic acid racemization in dentin is also possible for root dentin, apparently with good results. This is important particularly in those cases where a large portion of the coronal dentin is absent, for instance following dental treatment. In the investigation of root dentin, regression equations specific for root dentin must be employed in the estimation of age at death. Corresponding equations for third molars were calculated.     

Aspartic acid racemization of root dentin used for dental age estimation in a Polish population sample

Precise age determination of unidentified bodies and human remains is one of the essential tasks of forensic science. The aim of this study was to assess the applicability of using the enantiomeric composition of aspartic acid racemization in root and crown dentin for dental age estimation using a Polish population sample. Coronal and root dentin from four teeth groups from the mandible were studied using gas chromatography with mass spectrometry. The results demonstrated a very high correlation between the chronological age and enantiomeric composition in both of the dentin samples. Individual linear equations of root dentin with correlation coefficients between 0.96 and 0.98 and a standard estimation error of ±2.95–4.84 years validated the application of aspartic acid racemization as a significant practical contribution to everyday forensic medical practice. Discrepancies in methodological aspects and modifications that simplify the protocol are presented.     

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

Summary 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 using aspartic amino acid racemization from a femur

Age estimation is an important step in post-mortem investigation, and the aspartic acid racemization (AAR) of dentin is the best tool for age estimation from human remains. However, teeth may not be found or sufficiently preserved to analyse the Dextro/Levo (D/L) ratio, and so age estimation using AAR from other organs, such as bones, is required. The current study evaluated age estimation methods using aspartic amino acid racemization from a femur in a Thai population. D/L ratio analysis using high-performance liquid chromatography (HPLC) was performed on 40 femoral bones from 24 males and 16 females, and the correlation between D/L ratio and age was 0.8316. The correlation coefficient in the male sample was greater than the female samples – 0.912 and 0.716, respectively. The standard error of estimation of all samples was 11.01 years. The correlation between the D/L ratio and the age of the total amino acid fraction from a femur was not as strong as from dentin and single protein purification, such as elastin and osteocalcin, but this method was necessary because teeth may not be found with body remains, and this method is not too complicated for routine forensic work.     

Racemization in enamel among different types of teeth from the same individual

We measured the quantity of D-aspartic acid (degree of racemization of aspartic acid) in the enamel of different types of teeth from the same individual. We studied the correlation between the degree of racemization and the time of formation of each particular tooth, as well as the applicability of the degree of racemization to estimation of chronological age. If the environmental condition of the teeth is the same, the degree of racemization is expected to be highest in teeth that completed formation in the earliest period of time. Different degrees of racemization in enamel were found among different types of teeth, even in the same individual. The degree of racemization in enamel was found to be higher in molars than in incisors, and showed a tendency that did not necessarily coincide with the time of formation. This seemed to be due to the fact that the environmental temperature was higher in the molar region located deeper in the oral cavity than the front region, and that enamel was more affected by breathing air than dentin because the D/L ratios in enamel were lower than those in dentin. Using enamel, a better estimation of chronological age was obtained from calculations based on the degree of racemization of each type of tooth than from all the different teeth together. However, these estimated ages were not better than those from dentin.     

Age estimation: The state of the art in relation to the specific demands of forensic practise

Age estimation in cadavers, human remains and living individuals may clarify issues with significant legal and social ramifications for individuals as well as for the community. In such cases methods for estimating age should fulfil the following specific demands: (1) they must have been presented to the scientific community, as a rule by publication in peer-reviewed journals, (2) clear information concerning accuracy of age estimation by the method should be available, (3) the methods need to be sufficiently accurate and (4) in cases of age estimation in living individuals principles of medical ethics and legal regulations have to be considered. We have identified and summarized the methods that essentially fulfil these specific demands. In childhood and adolescence morphological methods based on the radiological examination of dental and skeletal development are to be recommended. In adulthood, the accuracy of most morphological methods is much reduced. Here a biochemical method based on aspartic acid racemization in dentine provides the most accurate estimates of age, followed by special morphological dental and skeletal methods. The choice of method has to take account of the individual circumstances of each case. Most methods require either the consultation of specialised and trained scientists or an adequate calibration by the “user”. Very few attempts have been made to find common standardisation, calibration and evaluation procedures or to develop means of quality assurance for methods of age estimation. Efforts in these directions are necessary to guarantee quality standards and adequate answers to the important legal and social issue of age estimation in forensic medicine. Received: 26 January 1999 / Received in revised form: 12 April 1999     

Biochemische Altersschätzung Zur Frage genetischer und soziokultureller (ethnischer) Einflüsse auf die Razemisierung von Asparaginsäure in Dentin

In an increasing number of cases, age estimation must be performed in individuals who do not correspond to the reference population with regard to genetic and/or cultural origin. Theoretically, this may result in errors in the biochemical age estimation based on aspartic acid racemization in dentine, if genetic and/or cultural influences cause variations in the protein composition of dentine. To clarify the question whether such influences have to be considered, teeth from Turkish and German individuals with known ages were analysed to achieve a direct comparison. The relations between aspartic acid racemization in dentine and age were identical in the two groups. Considering these data and the literature, there is no indication of genetic and/or cultural influences on the protein composition of dentine and on the results of age estimation based on aspartic acid racemization in dentine.     

Analysis of advanced glycation end products (AGEs) in dentine: useful for age estimation?

Ageing of the human organism results in the accumulation of modified molecules. Some of these molecular changes may be used for age estimation, as already shown for aspartic acid racemization (AAR). Another example for an accumulation of damaged molecules is advanced glycation end products (AGEs). We examined, (1) if the correlation between the concentration of AGEs (pentosidine) in root dentine and age is close enough to be used as basis for age estimation, and (2) if the combined analysis of AGEs and AAR in dentine may be a useful approach to rule out or to detect relevant effects of confounding factors in age estimation. We determined the pentosidine content of root dentine samples of 64 healthy teeth as well as in carious, “pink”, diabetic and heated teeth, and in teeth after different storage times. In 23 teeth, the extent of aspartic acid racemization (AAR) was determined in parallel. We observed a close relationship between the concentration of pentosidine in dentine and chronological age (r = 0.94) in healthy teeth. The analysis of pentosidine in dentine can theoretically be used as a basis for age estimation in healthy teeth of non-diabetic individuals; diabetic individuals may exhibit very high pentosidine levels in dentine. This finding limits the application of this method, since information regarding the question if an unidentified person suffered from diabetes mellitus or not are missing in most cases. Moreover, the method is not suitable to identify or rule out the influence of confounding factors in age estimation based on AAR, since both methods are sensible to the most relevant confounding factors (caries, heat).

     

Estimation of chronologic age using the aspartic acid racemization method. I. On human rib cartilage

Determinations of chronologic age are of great importance in forensic science. At present the aspartic acid racemization method on teeth provides one of the best means in adult individuals. However, if teeth are not available, some other stable tissue has to be used. In this study, the applicability of cartilage from the ribs has been tested. Specimens of rib cartilage were obtained at autopsy from 24 individuals aged 0.2–96 years. An acid-soluble peptide (SP) fraction and an insoluble collagen-rich (IC) fraction were prepared, and the ratio of D/L aspartic acid was determined using the HPLC technique. The correlation coefficient between the D/L ratio and age was r = 0.91 in SP and r = 0.97 in IC. It thus seems as if cartilage from non-weight-bearing areas may be a useful source of tissue for the estimation of chronologic age with the aspartic acid racemization method when teeth are not available.This study was supported by grant # B93-241-10277-01 from the Swedish Medical Research Council and Deutscher Akademischer Austauschdienst grant # 517 023 502 3     

Reliability of aspartic acid racemization rate for chronological age estimation—a systematic review and meta-analysis

Objective

The biochemical approach of dental age using aspartic acid racemization has been widely reported. The aim of this systematic review was to determine the reliability and accuracy of dental age estimation using aspartic acid racemization rate analysis.

Design

Eight research databases (PubMed, Google Scholar, Embase, Cochrane, MEDLINE, TRIP, Web of Science, and Scopus) were utilised to gather and assess published literatures in compliance with the PRISMA 2020 guidelines and reported in PROSPERO (CRD42020208877). This systematic review and meta-analysis focused solely on cross-sectional studies. The quality evaluation was performed using the GRADE system. The standardized mean difference between estimated and chronological age was meta-analyzed using the random effects model.

Result

The literature review yielded 213 studies, of which 26 were considered acceptable for inclusion in this report. Out of 26 studies, 19 presented sufficient evidence for meta-analysis and the remaining 7 were used to construct a qualitative review. According to the meta-analysis, premolar-related studies had the least variability (Tau² = 0.23; I² = 72%) among other tooth groups.

Conclusion

A substantial degree of heterogeneity was found in every type of tooth. Only the premolar teeth had lower degree variability; thus, it is safe to believe that the premolar tooth is the best for this type of age estimation. It is recommended to develop population-specific mathematical equations to improve the accuracy of this age estimation approach.

     

Age estimation based on aspartic acid racemization in elastin from the yellow ligaments

The yellow ligaments of the spine are characterized by an exceptionally high content of elastin, a protein with a proved longevity in several human tissues. This unique biochemical composition suggested a suitability of yellow ligaments for age estimation based on aspartic acid racemization (AAR), which was tested by determination of AAR in total tissue specimens and in purified elastin from yellow ligaments of individuals of known age. AAR was found to increase with age in both sample sets. The purified elastin samples exhibited a much faster kinetics than the total tissue, with ca. 3.7-4.6-fold higher apparent rates. The relationship between AAR and age was much closer in the purified elastin samples ( r=0.96-0.99) and it can therefore be used as a basis for biochemical age estimation. The analysis of total tissue samples cannot be recommended since the AAR values can be strongly influenced even by slight, histologically non-detectable variations in the collagen content. Age estimation based on AAR in purified elastin from yellow ligaments may be a valuable additional tool in the identification of unidentified cadavers, especially in cases where other methods cannot be applied (e.g. no available teeth, body parts).     

Deoxypyridinoline crosslinks in human dentin and estimation of age

A new biochemical method for estimation of dental age from human dentin is presented. An adaptation and modification of a dentin protein extraction protocol was developed by which deoxypyridinoline crosslinks were measured using an enzyme immunoassay technique. Deoxypyridinoline, a non-reducible collagen crosslink, was measured in human dentin samples from permanent individual molars. The measurement of this protein in dentin samples from 22 patients with ages ranging from 15 to 73 years old was carried out to obtain an estimate of age at death. By linear regression between deoxypyridinoline values and age, equations for age calculations were computed. The error between the estimated age and the known age at extraction was calculated. Our results show that this method produces an expected associated error on calibration averaging 14.9 years about the mean estimated values, at a 65% level of confidence. Values of sensitivities and specificities were calculated for deoxypyridinoline at different ages. We conclude that determination of deoxypyridinoline in human dentin extracts is a new and reliable procedure, potentially suitable for most forensic laboratories and which could be a useful tool to estimate human dental age. This method could be considered as an alternative method to calculate dental age, contributing to a more accurate estimation of chronological age when used in addition to other morphological or biochemical dental methods. Received: 15 May 1998 / Received in revised form: 16 October 1998     

Comparison of racemization rates between vital and endodontically treated teeth for age estimation

Amino acid racemization of dentin (AAR) is among the most precise methods for age estimation in unidentified adult cadavers. Although vital teeth are generally used for this technique, cases often have endodontically treated (Endo) teeth only. Therefore, the aim of this preliminary pilot study was to determine the applicability of Endo teeth by comparing AAR rates between vital and Endo teeth. Thirty-six dentin samples from vital teeth and 18 dentin samples from Endo teeth were analyzed, including the maxillary first premolar, maxillary second molar, and mandibular second premolar. Standard calibration curves for the mandibular second premolar and maxillary second molar were compared among vital teeth, Endo teeth, and mixed vital/Endo teeth. Discrepancies between estimated and actual ages were assessed by analyzing the AAR rates for the maxillary first premolar between vital and Endo teeth. The AAR rates for Endo teeth were higher than those for vital teeth in both the mandibular second premolar and maxillary second molar, and the correlation of the standard calibration curve for vital teeth only was highest (r = 0.982836; r = 0.92011467), followed by mixed (r = 0.949579; r = 0.76158) and Endo teeth only (r = 0.896082; r = 0.744991). In the maxillary first premolar, discrepancies were more than 10 years for all 5 Endo teeth, based on the standard calibration curve of vital teeth. The present study suggests that the AAR rates for Endo teeth might be affected by variation in dentin structure between vital and Endo teeth.     

Technical notes for age estimation using the femur: influence of various analytical conditions on D-aspartic acid contents

In order to make a better estimate of the individual age using bone samples, we evaluated the effects of various criteria on the analytical measurement of D-aspartic acid contents. Using compact bone from a male femur, we varied six analytical conditions (sample volume, sample particle size, hydrolysis temperature, hydrolysis time, hydrochloric acid volume during hydrolysis, and hydrochloric acid concentration during hydrolysis). D-form/L-form ratios were affected most by hydrolysis temperature (estimated age differences were 3.03 years/ degrees C), followed in order by hydrochloric acid volume (1.44 years/ml) and hydrochloric acid concentration (0.69 years/0.1 M). Larger sample particle sizes and hydrochloric acid volumes during hydrolysis tended to result in lower racemization rates. Nonetheless, within a range of 5-50 mg, sampling volume did not affect the detected D-aspartic acid contents. Since the racemization reaction rate in femur compact bone is slower than in dentin, bone samples seem to be more greatly influenced by analytic conditions than dentin. Tests must therefore be performed with caution, especially with regard to the hydrolysis temperature, hydrochloric acid volume and concentration when estimating age using femur samples.     

Concentration of collagen cross-links in human dentin bears no relation to the individual age

Lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) are collagen cross-link residues. HP is present in most tissues, whereas LP is specific for bone and dentin. Based on the current literature there are certain indications that measurement of the concentrations of HP and LP in dentin may be a valuable tool to determine the individual age. The purpose of this investigation was to assess if the concentrations of LP and HP in dentin increase during lifetime. We have investigated 173 molars from 173 individuals (2 through 78 years of age, 31 primary and 142 secondary teeth) in the course of the present study. Levels of LP and HP were measured by HPLC and fluorescence detection. The results show that dentinal concentrations of HP and LP did not increase with age and varied between individuals of the same age and that determination of dentinal concentrations of HP and LP cannot be used to determine the individual age.     

Differences in the D/L aspartic acid ratios in dentin among different types of teeth from the same individual and estimated age

We have studied the correlation between the level of D-aspartic acid in dentin and the period of dentin formation in different types of teeth from the same individual. Except for the third molar, it is generally agreed that the formation of permanent dentin follows the pattern of growth, occurring earliest in the first molar and last in the second molar. In middle-aged individuals, racemization among the different types of teeth was highest in the first molar, corresponding to the earliest period of dentin formation. In elderly individuals, however, racemization tended to be highest in the second molar, in which dentin is formed last during growth. It is assumed that this may be due to the higher ambient temperature of the second molars, which are located deeper in the oral cavity. Thus, these results suggest that in elderly individuals racemization in teeth that have been situated deep in the oral cavity for a long time is more influenced by the environment than by the period of tooth formation.     

Estimation of age at death based on aspartic acid racemization in elastic cartilage of the epiglottis

Age estimation based on aspartic acid racemization (AAR) has been applied successfully to various tissues. For routine uses, AAR is analyzed in dentine. For cases in which teeth are unavailable, analyzing AAR in purified elastin has been shown to be an alternative method. The suitability of elastic cartilage from the epiglottis as an elastin source for age estimation based on AAR was tested. A total of 65 tissue samples (cartilage) of epiglottis and 45 samples of elastin purified from the elastic cartilage of epiglottis samples were analyzed. While the d-aspartic acid content of total tissue samples increased with age only slowly, its increase with age in purified elastin samples was similar to that in purified elastin from other tissues. The relationship between the d-aspartic acid content and age was shown to be close enough for age estimation based on AAR in purified elastin from the elastic cartilage of the epiglottis, provided a sufficient quality of elastin purification. Age estimation based on AAR in purified elastin from the epiglottis might serve as a valuable alternative in cases in which other tissues (e.g., teeth) are unavailable.     

Estimation of chronologic age using the aspartic acid racemization method. II. On human cortical bone

Over the last 20 years a new chemical method, base on the racemization of aspartic acid, has been developed to be used for the estimation of chronologic age in adult individuals. The method has a good accuracy when used on dental enamel, dentine and cartilage. However, in forensic and archeological cases teeth and cartilage are not always available. Since preliminary studies have shown that there are some age-related changes of the D/L aspartic acid ratio also in bone, this study was carried out to further explore if the method could be used for age estimations of bone. Bone samples from 24 individuals, aged 0.2 to 95.6 years were analysed for the D/L ratios with HPLC-technique. Two different fractions of the bone were examined, an acid-soluble peptide fraction and an acid-insoluble collagen-rich fraction. The analyses showed age-related racemizations in both fractions, although of different rates. The correlation coefficients with age were 0.72 in the peptide fraction, and 0.84 in the collagen-rich fraction. It thus seems as if bone maybe used for age estimations when more stable tissues like dentine and cartilage are not available.This study was supported by grant # B93-241-10277-01 from the Swedish Medical Research Council and from Deutscher Akademischer Austauschdienst grant # 517 023 502 3     

Aspartic acid racemisation in purified elastin from arteries as basis for age estimation

Aspartic acid racemisation (AAR) results in an age-dependent accumulation of d-aspartic acid in durable human proteins and can be used as a basis for age estimation. Routinely, age estimation based on AAR is performed by analysis of dentine. However, in forensic practise, teeth are not always available. Non-dental tissues for age estimation may be suitable for age estimation based on AAR if they contain durable proteins that can be purified and analysed. Elastin is such a durable protein. To clarify if purified elastin from arteries is a suitable sample for biochemical age estimation, AAR was determined in purified elastin from arteries from individuals of known age (n = 68 individuals, including n = 15 putrefied corpses), considering the influence of different stages of atherosclerosis and putrefaction on the AAR values. AAR was found to increase with age. The relationship between AAR and age was good enough to serve as basis for age estimation, but worse than known from dentinal proteins. Intravital and post-mortem degradation of elastin may have a moderate effect on the AAR values. Age estimation based on AAR in purified elastin from arteries may be a valuable additional tool in the identification of unidentified cadavers, especially in cases where other methods cannot be applied (e.g., no available teeth and body parts).     

酸蚀处理时间对可乐丽菲露粘接强度影响的研究

目的研究37％磷酸酸蚀处理时间对可乐丽菲露树脂水门汀与牙本质粘接强度的影响。方法选取新鲜拔除的无龋坏人后牙20颗,分别制备出80个牙本质粘接面试件,随机分成5组,每组16个试件,并分别采取的不同酸蚀时间：0、10、20、30、40s,然后按可乐丽菲露的使用说明进行粘接,在37℃的生理盐水保存24h后,分别进行牙本质粘结抗剪切强度测试。结果在使用可乐丽菲露树脂水门汀进行牙本质粘接时,酸蚀牙本质粘结面10s可取得最高的牙本质粘接强度（24．62±3．60）Mpa（P〈0．05）。结论酸蚀处理可以显著提高可乐丽菲露树脂水门汀与牙本质的粘接强度。