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.     

Measuring Children's Suggestibility in Forensic Interviews

According to the scientific literature, childrens’ cognitive development is not complete until adolescence. Therefore, the problems inherent in children serving as witnesses are crucial. In preschool‐aged children, false memories may be identified because of misinformation and insight bias. Additionally, they are susceptible of suggestions. The aim of this study was to verify the levels of suggestibility in children between three and 5 years of age. Ninety‐two children were examined (44 male, 48 female; M = 4.5 years, SD = 9.62). We used the correlation coefficient (Pearson's r) and the averages variance by SPSS statistical program. The results concluded that: younger children are almost always more susceptible to suggestibility. The dimension of immediate recall was negatively correlates with that of total suggestibility (r = ?0.357 p < 0.001). Social compliance and source monitoring errors contribute to patterns of suggestibility, because older children shift their answers more often (r = 0.394 p < 0.001). Younger children change their answers more times (r = ?0.395 p < 0.001).     

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.     

Estimating Time Since Death from Postmortem Human Gut Microbial Communities

Postmortem succession of human‐associated microbial communities (“human microbiome”) has been suggested as a possible method for estimating postmortem interval (PMI) for forensic analyses. Here we evaluate human gut bacterial populations to determine quantifiable, time‐dependent changes postmortem. Gut microflora were repeatedly sampled from the proximal large intestine of 12 deceased human individuals as they decayed under environmental conditions. Three intestinal bacterial genera were quantified by quantitative PCR (qPCR) using group‐specific primers targeting 16S rRNA genes. Bacteroides and Lactobacillus relative abundances declined exponentially with increasing PMI at rates of N_t = 0.977e^?0.0144t (r² = 0.537, p < 0.001) and N_t = 0.019e^?0.0087t (r² = 0.396, p < 0.001), respectively, where N_t is relative abundance at time (t) in cumulative degree hours. Bifidobacterium relative abundances did not change significantly: N_t = 0.003e^?0.002t (r² = 0.033, p = 0.284). Therefore, Bacteroides and Lactobacillus abundances could be used as quantitative indicators of PMI.     

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.     

Secular Trend in the Development of Permanent Teeth in a Population of Istria and the Littoral Region of Croatia

The study evaluated secular trends in dental development during a period of 30 years, correlation between dental and chronological age in Istria and the littoral region of Croatia. The sample consisted of 1000 panoramic radiographs of children, aged 6–16 years (mean 10.0 ± 1.8), taken in the period 1977–1979 (N = 500; 243 females) and 2007–2009 (N = 500; 299 females). Dental age was assessed according to Demirjian's method. Correlation between chronological and dental age was linear, positive, high, and statistically significant in both periods and genders, ranging from 0.73 to 0.86. Dental age was underestimated when compared to chronological age by 1 year on average, more 30 years ago (?1.35 ± 1.17) than today (?0.63 ± 1.09), less for girls (?0.80 ± 1.22) than boys (?1.21 ± 1.10). A statistically significant positive secular trend in acceleration of dental development was present of 0.72 years during the 30‐year period and was more significant in girls than boys (0.83‐ and 0.51‐year acceleration).     

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.     

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.     

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 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.     

Epiphyseal Union of the Cervical Vertebral Centra: Its Relationship to Skeletal Age and Maturation of Thoracic Vertebral Centra

Epiphyseal union stages for cervical vertebral centra (ring epiphyses) were documented for 55 individuals (females and males, ages 14–27 years) from the Terry Collection, using the Albert and Maples method 1, to examine both its relationship to age at death and to thoracic data collected from the same individuals using the same method. Results showed a moderate correlation between cervical ring union and age (r = 0.63, p = 0.000), and a fairly low correlation between cervical and thoracic ring union (r = 0.41, p = 0.002). Paired samples t‐tests yielded a statistically significant difference between cervical and thoracic union mean values (p = 0.01). Union progressed earlier in cervical vertebrae and in females. Results indicated fairly substantial variation in both sexes. Findings may serve as a basic guideline for estimating a general age range at death for unknown skeletal remains and to corroborate findings from other skeletal age indicators.     

Determination of Sex: Interest of Frontal Sinus 3D Reconstructions

Frontal sinuses (FSs) have been studied in radiology, anthropology, and forensic anthropology. This study aimed to determine whether it was possible to predict the age and sex of an individual using FS volume. Sixty‐nine anonymized CT scans were imported to MIMICS 10.01^® software (Materialise N.V.), and each FS volume was calculated in mm³. There was an absence of significant difference between right and left FS volume (p = 0.173) and an absence of correlation between age and FS volume (Pearson's test; p = 0.705). Sexual dimorphism was significantly different (p = 0.001). However, the most discriminant datum for determining sex was found to be the total FS volume (sum of an individual's right and left FS volumes) with linear discriminant Fisher's function coefficients of ?2.759 for the male group and ?1.275 for the female group. With this model, 72.5% of our sample was correctly classified according to sex.     

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.     

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.     

Dental Age Estimation: A Test of the Reliability of Correctly Identifying a Subject Over 18 Years of Age Using the Gold Standard of Chronological Age as the Comparator

This study was designed to test the reliability of using the third molars to demarcate between child and adult status. A total of 2000 dental panoramic tomographs were used for assessment of the calculated age using the 8‐stage system of tooth development and applied to all four third molars. The LL8 was also assessed using this 8‐stage system. For each tooth development stage, the Normal distribution and percentile summary data were estimated. The calculated dental age compared with the chronological age was statistically significantly different (p < 0.001) for both females and males giving underestimates of the true age. Comparison of single tooth dental age and chronological age was only slightly different. The most important finding is that the assignment to above or below the 18‐year threshold, in the age range 17 years to 19 years, could be wrong on up to 50% of occasions.     

Comparative Analysis of Insect Succession Data from Victoria (Australia) Using Summary Statistics versus Preceding Mean Ambient Temperature Models

Minimum postmortem interval (mPMI) can be estimated with preceding mean ambient temperature models that predict carrion taxon pre‐appearance interval. But accuracy has not been compared with using summary statistics (mean ± SD of taxon arrival/departure day, range, 95% CI). This study collected succession data from ten experimental and five control (infrequently sampled) pig carcasses over two summers (n = 2 experimental, n = 1 control per placement date). Linear and exponential preceding mean ambient temperature models for appearance and departure times were constructed for 17 taxa/developmental stages. There was minimal difference in linear or exponential model success, although arrival models were more often significant: 65% of linear arrival (r² = 0.09–0.79) and exponential arrival models (r² = 0.05–81.0) were significant, and 35% of linear departure (r² = 0.0–0.71) and exponential departure models (r² = 0.0–0.72) were significant. Performance of models and summary statistics for estimating mPMI was compared in two forensic cases. Only summary statistics produced accurate mPMI estimates.     

Homicide Rates in Lithuania

Homicide rate in Lithuania between 2004 and 2013 decreased and reached an average of 6.7 per 100,000 people in 2013. The data regarding forensic autopsies of intentional homicide victims were obtained from the State Forensic Medicine Service. Spearman's correlation test was used to assess trends in the homicide rates. A significant correlation was observed between homicide distribution and the following variables: Lithuania's gross domestic product (r = ?0.85, p = 0.003), the number of alcohol intoxication cases of victims (r = 0.97, p < 0.05). After regression model adjustments, these variables remained significantly associated with the homicide distribution (p < 0.05). 73% of victims were men, with a mean age of 45.5 ± 15. Alcohol intoxication was present in 58% of victims. 66% of homicides were carried out indoors, 57% in urban area. The presented findings help decide which prevention programs may be the most effective in homicide rate reduction.     

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.     

Does the Prior Application of the Field Kit Bullet Hole Testing Kit 3 on a Suspected Bullet Hole Bias the Analysis of Atomic Absorption Spectrophotometry?

Forensic ballistics is the study of bullet trajectory and consists of determining gunshot residue (GSR) to identify bullet holes. Among several highly sensitive methods, atomic absorption spectrophotometry (AAS) is employed to analyze GSR in the laboratory. However, it is sometimes necessary to identify bullet holes immediately at a crime scene. The purpose of this examination was to determine whether the use of the field test Bullet Hole Testing Kit 3 (BTK3) on a suspected bullet hole would influence the outcome of AAS‐analysis: Three commonly encountered firearms (Glock17, Tokarev, and Colt) were fired at skin, wood, and cloth. AAS‐analysis was performed with and without previous BTK3 application. The results clearly indicate that there is no significant interaction on the grounds of BTK3 use (BTK3 vs. no‐BTK3 [kit_nokit] [Pb: p = 0.1309; Sb: p = 0.9111], material*kit_nokit [Pb: p = 0.5960; Sb: p = 0.9930], distance*kit_nokit [Pb: p = 0.4014; Sb: p = 0.9184], and firearm type*kit_nokit [Pb: p = 0.9662; Sb: p = 0.9885]); hence, applying this field kit does not falsify later AAS outcomes.     

The Reliability of the Greulich and Pyle Atlas When Applied to a Modern Scottish Population

This study examines the reliability of age estimation utilizing the Greulich and Pyle atlas in relation to a modern Scottish population. A total of 406 left‐hand/wrist radiographs (157 females and 249 males) were age‐assessed using the Greulich and Pyle atlas. Analysis showed that there was a strong correlation between chronological age and estimated age (females R² = 0.939, males R² = 0.940). When age groups were broken down into year cohorts, the atlas over‐aged females from birth until 13 years of age. The pattern for males showed that the atlas under‐estimated age until 13 years of age after which point it consistently over‐aged boys between 13 and 17 years of age. This study showed that the Greulich and Pyle atlas can be applied to a modern population but would recommend that any analysis takes into account the potential for over‐ and under‐aging shown in this study.