Age estimation in forensic sciences: application of combined aspartic acid racemization and radiocarbon analysis.

Age determination of unknown human bodies is important in the setting of a crime investigation or a mass disaster because the age at death, birth date, and year of death as well as gender can guide investigators to the correct identity among a large number of possible matches. Traditional morphological methods used by anthropologists to determine age are often imprecise, whereas chemical analysis of tooth dentin, such as aspartic acid racemization, has shown reproducible and more precise results. In this study, we analyzed teeth from Swedish individuals using both aspartic acid racemization and radiocarbon methodologies. The rationale behind using radiocarbon analysis is that aboveground testing of nuclear weapons during the cold war (1955-1963) caused an extreme increase in global levels of carbon-14 ((14)C), which has been carefully recorded over time. Forty-four teeth from 41 individuals were analyzed using aspartic acid racemization analysis of tooth crown dentin or radiocarbon analysis of enamel, and 10 of these were split and subjected to both radiocarbon and racemization analysis. Combined analysis showed that the two methods correlated well (R(2) = 0.66, p < 0.05). Radiocarbon analysis showed an excellent precision with an overall absolute error of 1.0 +/- 0.6 years. Aspartic acid racemization also showed a good precision with an overall absolute error of 5.4 +/- 4.2 years. Whereas radiocarbon analysis gives an estimated year of birth, racemization analysis indicates the chronological age of the individual at the time of death. We show how these methods in combination can also assist in the estimation of date of death of an unidentified victim. This strategy can be of significant assistance in forensic casework involving dead victim identification.

Preparation of dentin standard samples for age estimation based on increased aspartic acid racemization rate by heating.

Age estimation in adults based on aspartic acid racemization (AAR) provides fewer errors and higher precision than that based on bone morphology for the identification of cadavers. The technique has been established in some labs as a routine method. However, as the essential requisites for the technique, a wide age range of teeth of the same type as the target tooth must be collected for calibration for each examination. We investigated whether dentin standard samples could be prepared by increasing the AAR rate via heat. Powdered dentin was prepared from a maxillary first premolar (13 years) and heated for 0-72 h at 110 °C. The extent of AAR increased significantly with heating time and the correlation was strong (r = 0.913; p < 0.01). Similar results were found for a mandibular canine (24 years, r = 0.948; p < 0.01) and a maxillary third molar (20 years, r = 0.944; p < 0.01). We attempted to estimate the age of four maxillary first premolars of persons aged 25-58 years by using the heated samples (18 years, 12 h to 7 days). The differences between the actual and estimated ages were within ±5 years. The stability of the AAR rates in the powdered dentin during storage at 22-25 °C, 4 °C, and -30 °C was examined after 1 year and no significant changes had occurred. We were able to prepare dentin standard samples and created a calibration curve. This is a pilot study that needs to be validated before it can be used in forensic practice.

Regional differences in D/L aspartic acid ratios in the human mandible as a possible indicator of the bone remodeling rate.

In slowly metabolizing tissue such as tooth, bone and ocular lens, D-amino acids converted from L-amino acids accumulate with age and thus reflect the tissue turnover rate. To investigate whether D-amino acids play a role in determining the bone remodeling rate, we measured the accumulation of D-aspartic acid, which has the fastest rate of racemization, in various areas of the mandible. The level of D-aspartic acid was higher in the ramus than in the body (P < 0.01), and within the body, the level was higher in the basal area than in the alveolar area (P < 0.01). Within the alveolar area, the level of D-aspartic acid was higher in the molar region than in the incisal region (P: 0.05-0.01). No correlation was found between the accumulated level of D-aspartic acid with age, because all the specimens were obtained from elderly people with only a few years difference in age. There was also no correlation between D-aspartic acid and sex. In conclusion, we suggest that accumulation of D-aspartic acid in the mandibular bone reflects the differences in remodeling associated with occlusion. The incisal portion of the alveolar area of the mandible (above the mandibular canal), shows the greatest evidence of active remodeling.

Comparison of aspartic acid racemization between mammoth and human dentinal tissues.

OBJECTIVE: Comprehensive research has revealed the complexity and heterogeneity of amino acid racemization (AAR) in biological samples. Therefore, to date, inherent uncertainties in relative dating as well as in determination of age at death have highlighted the significance of further investigations to explore aspartic acid kinetics in biominerals. Here we present data on an archaeological sample obtained from a mammoth tusk, in comparison to a previously well-described biomineral, to modern human dentin. DESIGN: Since no experimental investigations were carried out on this topic earlier, we provide the first estimates on racemization kinetics and time dependent tendencies in mammoth dentin. We analysed our samples using a protocol, which has been adopted for tooth dentin investigation. We measured different racemization rates amongst mammoth dentin layers. RESULTS AND CONCLUSIONS: Significantly, an overall low racemization rate was detected compared to human dentin. Kinetic experiments were performed by heating the samples, and linear correlations were established between racemization rates and heating time. The results allow us to derive racemization rate constants and activation energy for both samples. Extrapolation for ambient temperature showed increased velocity in mammoth dentin, which can be explained by biodeterioration of ancient collagen and by diagenetic changes in amino acid composition influenced by environmental conditions.

Age-dependent changes in the racemisation ratio of aspartic acid in human alveolar bone.

We investigated the racemisation ratio of aspartic acid (Asp) in alveolar bone. In addition, we designed and created a new column to detect Asp in a short period of time, which allowed us to detect d-Asp and l-Asp separately from each other within 5min. Comparing identical ages, the racemisation ratio of alveolar bone was generally lower than that of other bones reported so far. This result suggests that alveolar bone is metabolically more active than other bones, as expected. The rate constant for the racemisation reaction (k(y)) of alveolar bone was calculated to be 0.000338 in males and 0.000084 in females. The rate constants in males and females were each similar to the respective ratios of the femur. This result suggests that the age-dependent reduction in metabolic turnover in alveolar bone proceeds similarly to that in the femur, although those changes proceed more slowly in females than in males. The correlation coefficient between the racemisation ratio of alveolar bone and chronological age was 0.660. It was high in males (r=0.912) and low in females (r=0.527), and this gender difference was statistically significant (P: 0.01-0.001), as in the femur.

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.

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.

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 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.

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.

Age-related changes in the D-aspartic acid content of the cranial bones in senescence-accelerated mice (SAM).

We measured the age-related changes in the D/L-aspartic acid ratio (D/L ratio) of cranial bones in two different sublines of senescence-accelerated mice (SAM/Iw): SAMP2/Iw (SAM, prone 2/Iwate) and SAMR1/Iw (SAM, resistant 1/Iwate). In SAM/Iw under 9 months of age, the D/L ratio in SAMP2/Iw was higher than that in SAMR1/Iw (p<0.001). However, in all 12-month old SAM/Iw, whether SAMP2/Iw or SAMR1/Iw, the D/L ratio was higher in males than in females. There were minor differences in the age-related changes in the D/L ratios between different types of cranial bone. In 1-month old SAM/Iw the rectal temperature was similar in both males and females. However from the age of 2 months, there was a divergence, with the rectal temperature measurement in SAMP2/Iw reaching a peak 2 months of age, whereas SAMR1/Iw had the highest temperature readings at 6 months of age. In both SAMP2/Iw older than 2 months, and SAMR1/Iw older than 6 months, the temperature readings tended to gradually decrease with age in both males and females. These results suggest that the differences in D/L-aspartic acid ratios were dependent on the age, gender, and strain of SAM/Iw, but were not dependant on the type of cranial bone studied. It seems likely that these differences may be related mainly to changes in body temperature.

[Usefulness of postmortem dental panoramic X-ray photographs taken using a portable radiography apparatus for dental identification].

Fourteen unidentified human corpses were subjected to personal identification between 1999 and 2001. They included 5 drowning victims, three extensively burnt bodies, three skeletonized bodies, two highly putrefied bodies, and a body that had been run over by a train. Antemortem dental records were available in 12 cases, and all were positively identified. For 10 of these cases, antemortem panoramic X-ray photographs were obtained. For postmortem examination, panoramic X-ray photographs were taken using a portable dental panoramic radiography apparatus, LPX7007 (ASAHI ROENTGEN, Japan), during autopsy. Comparison of the ante- and postmortem panoramic X-ray photographs gave accurate and useful information not only about dental treatment but also the anatomical features surrounding the upper and lower jaws. This method is not time-consuming and also has the advantage of allowing dental treatment to be examined extraorally in cases where it is difficult to open the mouth of the corpse.

Estimation of chronological age from the racemization rate of L- and D-aspartic acid: how to completely separate enantiomers from dentin.

Estimation of chronological age is essential in forensic and archeological science. The racemization method is one of the best methods to meet the demands of these scientific fields, providing both accuracy of the estimated age and simplicity of technique. In general, living organs are composed of L-form amino acids. Conversion from L-form to D-form amino acids is a first-order chemical reaction. Thus, the quantity of D-form amino acids in an organ is proportional to the passed time (age) after organ completion if no protein turnover occurs after organization. However, every living organ undergoes some degree of protein turnover. Therefore, organs with low metabolic rates, such as teeth and bone, should be targeted for the racemization method. The most critical point of the technique may be the complete separation of D- and L-forms by gas chromatography because of the very small amounts of D-form amino acids present. We describe the detailed procedures and the critical points for obtaining reliable estimated ages using the racemization method.

Comparative analysis of aspartic acid racemization methods using whole-tooth and dentin samples.

One way to estimate biological age is to use the aspartic acid (Asp) racemization method. Although this method has been performed mostly using enamel and dentin, we investigated whether an entire tooth can be used for age estimation. This study used 12 pairs of canines extracted from both sides of the mandible of 12 individuals of known age. From each pair, one tooth was used as a dentin sample and the other as a whole-tooth sample. Amino acids were extracted from each sample, and the integrated peak areas of D-Asp and L-Asp were determined using a gas chromatograph/mass spectrometer. Statistical analysis was performed using the D/L-Asp ratio. Furthermore, teeth from two unidentified bodies, later identified as Japanese and Brazilian, were examined in the same manner. Results showed that the D/L ratios of whole-tooth samples were higher overall than those of dentin samples. The correlation coefficient between the D/L ratios of dentin samples and their age was r=0.98, and that of the whole-tooth samples was r=0.93. The difference between estimated age and actual chronological age was -0.116 and -6.86 years in the Japanese and Brazilian cases, respectively. The use of whole teeth makes the racemization technique easier and can standardize the sampling site. Additionally, using only a few tooth samples per analysis made it possible to reanalyze known-age samples. Although the difficulty in obtaining a proper control sample has prevented racemization from being widely used, the method described here not only ensures the availability of a control tooth, but also enables the teeth to be used for other purposes such as DNA analysis. The use of a whole tooth will increase the application of the racemization technique for age determination.

Comparison of age estimation in Japanese and Scandinavian teeth using amino acid racemization.

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.

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.

Age estimation by racemization method in teeth: application of aspartic acid, glutamate, and alanine.

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.

Age estimation by amino acid racemization in human teeth.

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.

Novel chiral selectors anchored on polydimethylsiloxane as stationary phases for separation of derivatized amino acid enantiomers by capillary gas chromatography.

Six chiral selectors of S-(-)-t-Leu-cyclopropylamide, S-(-)-t-Leu-cyclopentylamide, S-(-)-t-Leu-cyclohexylamide, S-(-)-t-Leu-cycloheptylamide, S-(-)-t-Leu-cyclooctylamide, S-(-)-t-Leu-cyclododecylamide have been prepared and anchored individually through amide bonding to a polydimethylsiloxane functionalized with 2,2,2-trifluoroethyl ester groups by way of nucleophilic displacement reaction. The resulting chiral polysiloxanes have been provided as stationary phases for the separation of amino acid enantiomers by capillary GC. Amino acids were derivatized into N(O)-trifluoroacetyl isopropyl esters. Especially, polydimethylsiloxane anchored with S-(-)-t-Leu-cyclooctylamide was found to be efficient for the separation of aspartic acid (Asp) enantiomers. The method was applied to the estimation of ages from the extent of Asp racemization in human dentines.

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.