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.     

Radiographic determination of developmental age in fetuses and stillborns.

The gestational age and/or viability of a fetus can become an important forensic issue. Several investigators have produced linear regression formulas based on crown-heel length (CHL), crown-rump length, or body diameters to determine gestational age. This study re-examines the relationship between fetal long bone length and CHL and tests a method of estimating CHL, and therefore gestational age, from radiographic measurements of the major long bone diaphyses. The results are compared with data based on dry bone measurements. Data from 252 cases confirm a strong correlation between all long bone lengths and CHL (> or = r = .9063; p < 0.01). Long bone length means for each CHL group are presented, as well as regression formulas for estimating CHL from radiographic measurements of the long bone diaphyses. The findings correspond closely with results based on a European sample, thereby validating that reference population as a normative sample for fetal analysis in the United States. The radiographic method can be used in instances where skeletal preparation is impossible or undesirable.     

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.     

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。     

Practical experiences with the determination of cadaver age by evaluation of bacterial metabolic products

In an experiment a corpse had been kept at room temperature (16 degrees-23 degrees C) for 1163 h. At regular intervals brain samples were taken and the content of free amino acids and related compounds was determined by column chromatography. It could be demonstrated that in a period of 4-20 days postmortem the age of the corpse could be calculated from the concentrations of alpha-aminobutyrate (ABU), gamma-aminobutyrate (GABA), and glutamic acid (GLU) in brain employing the previously [5] presented formula: (formula; see text) T is the postmortem time lapse (days). ABU, GABA, and GLU are the concentrations (mumol/g) wet tissue of the corresponding amino acids. During this postmortem interval there is nearly a linear correlation; from this interval a correct assignment between concentrations and time cannot be given. The determination method is in the range of about 15 degrees-25 degrees C (guarantees bacterial growth and metabolism) independent of ambient temperature. The brain samples (cortex or parts of the putrified brain mush) can be taken without any special precaution during normal autopsy. The results of the experiment (in Fig. 1) were supported by some practical cases where the time lapse since death was well (in Fig. 1) or reasonably (in Fig. 1) known. These results suggest that this method allows in many cases the determination of the age of a corpse found in a warm environment (approximately 15 degrees-25 degrees C) approximately 4-20 days after death.     

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.     

女性胸骨X线变化与年龄关系的研究

研究北京地区137例女性胸骨X线影像学变化，以其与年龄关系密切相关的影像学标志点作为变量，并依据各标志点在不同年龄阶段的变化，制定了各自的评分标准。用该标准观测全部样本，将所得数据与年龄一起输入电子计算机，建立了根据女性胸骨X线变化推算年龄的多元回归方程，即：Y=9．6641＋1．6999X（1）＋2．7486X（2）十1．8929X（3）＋1．5561X（4）＋0.4255X（5）。经方差分析F＞F0.01（5.131），回归高度显著。本研究所建立的预测年龄方程，在无名尸骨的法医学鉴定中具有实用价值。     

A method of age estimation using Raman microspectrometry imaging of the human dentin

The aim of this work is to establish a correlation between ageing and Raman spectra imaging of human teeth. Raman spectrum of an analysed sample is characteristic of its chemical components. By exploring the different dentinal areas on each tooth, we sought to find a correlation between noticeable variability on Raman spectra and the stages of dentinal evolution with advancing age. A study on 30 teeth analysed by Raman microspectrometry was used to select predictors of age, and it allowed us to determine a regression formula, with age as a dependent variable. Our data were computed by means of a statistical method called partial least squares (PLS) regression. This method was specially adapted for this kind of situation, where we have numerous predictors and relatively few observations. The regression formula was performed on a test sample of four more teeth, and we obtained a correct age estimation, with a mean error of more or less than 5 years. The main benefits of this method was a minimal and non-destructive tooth preparation, which led to an efficient age prediction, for any age group.     

The use of enzymatic hydrolysis for isolation of barbituric acid derivatives from blood (as exemplified by phenobarbital and barbamyl)

Modern isolation techniques by direct extraction with organic solvents or after protein precipitation by various sedimenting or salting-out agents are characterized by low efficiency and do not permit to liberate derivatives of barbituric acid from their complexes with blood proteins. The use of enzymatic hydrolysis makes it possible to break bonds between barbiturates and protein and thereby improve the efficiency of isolation. We performed enzymatic hydrolysis of the model phenobarbital-blood and barbamyl-blood complexes with the use of trypsin, pepsin, chymotrypsin, and papain. The degree of phenobarbital extraction with trypsin and barbamyl was estimated at 62.1 +/- 1.2% and 75.1 +/- 1.6% respectively; in other words, it was 32.7 +/- 1.0% and 51.1 +/- 1.0% higher than that achieved by traditional methods. Certain validation characteristics of the new method are presented.     

Application of Kvaal et al.'s age estimation method to panoramic radiographs from Turkish individuals

Age estimation of living adult individuals can be accomplished with limited methods. Radiographic dental methods based on the pulpal narrowing with secondary dentin formation have been presented. In the present study, Kvaal et al.'s method, one of the radiographic dental age estimation methods, was applied to panoramic radiographs from Turkish individuals. The correlation between chronological and estimated ages was examined and the feasibility of length and width measurements of pulp cavity was evaluated for age estimation. The study population consisted of 123 patients with ages ranging from 14 to 57 years. The measurements of the length and width of six types of teeth on digitized panoramic radiographs were performed, and the ratios between tooth and pulp cavity measurements were calculated. Age was estimated using the linear regression models presented by Kvaal et al. and Paewinsky et al. High differences were observed between chronological and estimated ages. Measurement ratios showed no significant or weak correlation with age. The linear regression models were derived using variables that were significantly correlated with age. The determination coefficients of the models varied from 0.035 to 0.345. In conclusion, a difference of more than 12 years in the chronological and estimated ages derived using regression models in literature was found on panoramic radiographs in Turkish individuals. The length and width of the pulp cavity, measured according to the method of Kvaal et al. using panoramic radiographs, were insufficient to precisely estimate the age of Turkish individuals.     

Variations in pulp/tooth area ratio as an indicator of age: a preliminary study

This paper details a method for age determination of adults from single rooted teeth. The sample consisted of 100 Italian white Caucasian patients (46 men, 54 women) aged between 18 and 72 years. The single rooted maxillary right canine was utilized in this preliminary study. Pulp/root ration, tooth length, pulp/tooth length ratio, pulp/tooth area and pulp/root width ratios at three different levels were computed. Pearson's correlation coefficients between age and these variables showed that the ratio between pulp and tooth area correlated best with age (r2 = 0.85). Stepwise multiple regression models yielded a linear relationship between pulp/root width at mid-root level and chronological age and a linear relationship when pulp/tooth area was compared to age. Statistical analysis indicated that these two variables explain 84.9% of variations in estimated chronological age. The median of the absolute value of residual errors between actual and estimated ages was less than four years.     

Age estimation methods using anthropological parameters on human teeth-(A0736)

The research was conducted on the 160 intact extracted human teeth with one and two roots of the known age and sex. The teeth were disinfected, dried and X-rayed. After that the section of the longitudinal cut through the teeth was performed in order to facilitate monitoring of all tissues and morphological characteristics of the teeth. The age was determined in three ways: Method 1 [G.Bang, E. Ramm, Determination of age in humans from root dentin transparency, Acta Odontol. Scand. 28 (1970) 3-35]--analysis of the translucency of the root dentine, Method 2 [S. Kvaal, T. Solheim, A non-destructive dental method for age estimation, J. Forensic Odonto-stomatol. 12 (1994) 6-11]--analysis of the root and the root canal from the X-ray, Method 3 [G. Johanson, Age determination from human teeth, Odontol. Revy. 22 (1971) 1-126]--analysis of six parameters on each teeth. All data were subject to the correlation and regression analysis which showed the following: all of the three applied methods were in the significant correlation with the real age, and the best of them proved to be Method 3 where the coefficient of correlation was 0.85, p<0.001. The teeth of the maxilla are more convenient for the age determination than the teeth of mandible. They are in the significant strong correlation with the known real age, and in Method 3, the coefficient of correlation is 0.78, p<0.001. Age determination of the teeth with two roots is in significant correlation with the known real age p<0.001 in relation to the determined age on the teeth with one root. The results show that sex too, is in significant correlation with the real age, p<0.001. In practice, the methods used and the results achieved in this research have been enabling the dental age estimation of human remains from mass graves after the 1991 war in Croatia.     

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 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.     

基于紫外可见积分球反射光谱法鉴定血痕经历时间

目的建立一种快速鉴定血痕陈旧度的方法。方法在实验室条件(20℃、25℃、30℃)下,使用UV-2450型紫外可见分光光度计,利用反射附件ISR-240A积分球,以BaSO_4标准白板做参比,间隔一定时间测定健康人耳静脉血血痕纱布的反射光谱,采集特定波长反射率R_(541)、R_(577),计算R_(541)/R_(577)值,使用SPSS 17.0软件进行直线拟合与回归分析。结果一定条件下8 h内,血痕经历时间与其特定波长紫外可见反射率比值回归分析的R~2均大于0.8,回归方程成立,血痕经历时间与R_(541)/R_(577)值具有高度相关性。结论所建立的方法简便、快速、无损,可靠性好,可以对实验室条件下8 h内血痕经历时间进行鉴别。     

Non-destructive testing techniques for the forensic engineering investigation of reinforced concrete buildings

This study describes in detail the results of a laboratory investigation where the compressive strength of 150mm side-length cubes was evaluated. Non-destructive testing (NDT) was carried out using ultrasonic pulse velocity (UPV) and impact rebound hammer (IRH) techniques to establish a correlation with the compressive strengths of compression tests. To adapt the Schmidt hammer apparatus and the ultrasonic pulse velocity tester to the type of concrete used in Algeria, concrete mix proportions that are recommended by the Algerian code were chosen. The resulting correlation curve for each test is obtained by changing the level of compaction, water/cement ratio and concrete age of specimens. Unlike other works, the research highlights the significant effect of formwork material on surface hardness of concrete where two different mould materials for specimens were used (plastic and wood). A combined method for the above two tests, reveals an improvement in the strength estimation of concrete. The latter shows more improvement by including the concrete density. The resulting calibration curves for strength estimation were compared with others from previous published literature.     

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.     

GC/MS法联合分析生物检材中的尼美舒利、二氧丙嗪

目的建立生物检材中尼美舒利、二氧丙嗪的GC/MS检验方法。方法采用浸取法提取,GC/MS定性检验,内标法选择离子检测（SIM）模式定量检验。结果最低回收率为69%,尼美舒利的线性相关系数为0.998,克咳敏的线性相关系数为0.9973。结论该方法操作简便快速,提取率高,可用于实际案件中尼美舒利、二氧丙嗪的检验。     

人外周血白细胞端粒DNA长度与年龄的定量关系

目的探讨人外周血白细胞端粒DNA长度变化规律及其与年龄的关系,为法医学推断年龄提供理论依据。方法抽取西藏那曲地区0~81岁健康人外周血样本105例,其中男性53例,女性52例,采用Southern杂交法检测其端粒限制性片断平均长度。各年龄组性别差异作t检验,端粒长度与年龄的关系作相关及回归分析。结果人外周血白细胞端粒长度随年龄增长逐渐缩短,且存在性别差异;得到推断年龄回归方程:Y=-16.539X+236.287±9.832。结论人外周血白细胞端粒DNA长度与年龄存在明显相关性,为法医学在分子水平上推断个体年龄提供了可能。