Susumu Ohtani

Estimation of age from the teeth of unidentified corpses using the amino acid racemization method with reference to actual cases

We report the results of a study for estimating the age of unidentified corpses from teeth using the amino acid racemization method. By calculating the ratio of D-aspartic acid to L-aspartic acid (referred to as the D/L ratio), we studied samples from 24 corpses of known identity. The results showed that the approach yielded satisfactory age estimates with an accuracy of ± 3 years. Detailed examination of these results indicated that powdered dentin produced more favorable results than fragmented dentin. No differences in the estimated age related to cause of death were noted. However, better results were obtained with a larger number of control teeth and a higher correlation coefficient between the D/L ratio and the actual age of the control teeth. In terms of age and estimation accuracy, corpses of individuals >40 years of age generally gave more satisfactory results than those of younger individuals. It was also reconfirmed that preparation of control teeth is required each time an age estimate is performed.

Evaluation of aspartic acid racemization ratios in the human femur for age estimation.

Levels of D-aspartic acid (D/L ratio) in cranial non-collagen proteins (acid-soluble peptide fractions) have been reported to increase with age. We isolated total amino acid fractions from the femur and separately isolated acid-insoluble collagen fraction and acid-soluble peptide fractions; then D/L ratios were measured from each fraction by gas chromatography. We evaluated the applicability of their D/L ratios for age estimation based on their correlation coefficient. A sex-related difference was observed in the D/L ratio. In particular, aged females showed a low ratio, suggesting an association with bone disorders. In males, the D/L ratios of acid-soluble peptide fraction showed the highest correlation rate (r = 0.969) with age, and those of total amino acid fraction showed the highest correlation rate (r = 0.633) with age in females. Without separation of male and female, the D/L ratios of total amino acid fraction showed the highest value (r = 0.853). The D/L ratio of acid-soluble peptide fractions differed according to the size of bone powder particles, being higher for larger particle sizes. These results suggest that the application of D/L ratio from total amino acid fraction is the most effective method for estimating age using the human femur. However, care is necessary when studing cadavers that might be females.

Racemization of aspartic acid in human cementum with age.

This study explored the application of the racemization reaction to forensic dental medicine in relation to estimation of age from tooth cementum. Cementum from the cervical two-thirds and the apical one-third of 16 upper incisor teeth gave ratios of D-/L-aspartic acid that correlated highly with actual age, the correlation coefficients being r = 0.993-0.996. For whole cementum from lower central and lateral incisors, first and second premolars (n = 8) the correlation coefficients were r = 0.984-0.997. Incisors gave more reliable results than premolars. The study was extended to compare cementum, enamel and dentine from first premolar teeth of the same individuals. The correlation of the ratio of D-/L-aspartic acids with actual age was highest for dentine (r = 0.992), followed by cementum (r = 0.988) and enamel (r = 0.961). These results indicate that the racemization reaction in cementum proceeds in a constant manner, and confirm that cementum remains stable throughout the individuals life. Accurate estimation of age is thus possible when cementum is subjected to the amino-acid racemization method.

Estimation of Age from Dentin by Using the Racemization Reaction of Aspartic Acid

To confirm the usefulness of the whole dentin in longitudinal sections for estimating age using the racemization ratio (D/L ratio) of aspartic acid, we compared longitudinal and transverse sections prepared from bilateral teeth in the same jaw of the same subjects. The D/L ratio was measured by gas chromatography. A better correlation between the D/L ratio and true age was observed when using longitudinal sections (r = 0.995) than when using transverse sections (r = 0.984–0.987). In the low age group, the D/L ratio was high in the crown and decreased toward the root apex. In the middle-advanced age groups, however, the D/L ratio was high in the crown as well as the area close to the root apex. The racemization reaction rate was higher in longitudinal than in transverse sections. These results suggest that the whole dentin in longitudinal sections of the central area of the tooth should be examined for accurate estimation of age.

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.

Estimation of age from dentin by utilizing the racemization of aspartic acid: influence of pH

The rate of racemization of aspartic acid is considered to be influenced by environmental factors. The influence of pH (4 and 9) on teeth was investigated by a heating experiment for precise estimation of age at death. The rate of racemization of aspartic acid in dentin (D/L ratio) was determined by gas chromatography. The reaction rate was highest in dentin in a pH 9 environment, followed by that in water, that in a pH 4 environment, and that in a dry state. The age estimated from teeth left in these environmental states (16 degrees C) for 1 year showed only a slight increase of < 0.2 years for teeth in a dry state; a pH 4 environment and in water. However, the age estimated from teeth in the pH 9 environment showed an increase of 0.6 years and that from teeth left for 5 years showed an increase of 3.2 years. These results suggest that careful attention should be paid to age estimation from specimens left in an alkaline state for an extended period (several years). Previous studies on estimation of age from bones have-shown no influence of pH 4 or 9 on teeth. It is interesting to consider whether these conflicting findings are attributable to differences between experimental methods or differences in components between bone and tooth.

IMPROVEMENT OF AGE ESTIMATION USING AMINO ACID RACEMIZATION IN A CASE OF PINK TEETH

Age was estimated from pink teeth using racemization of dentinal aspartic acid. Materials for identification were two lower second premolars. The body was determined to be that of a 40-year-old man; however, the age of the decedent had been estimated to be 29 and 30 years by the conventional method and 30 years from findings in the oral cavity. To clarify the cause of this difference, the powdered teeth were further washed in 0.01 mol/L hydrochloric acid. The racemization ratio (D/L ratio) of ordinary white teeth from persons of known age was slightly lower than that before washing, whereas that of the teeth used for identification was higher than before washing. The calculated age of the decedent using the racemization ratio of his teeth was between 36 and 37 years. These results suggest that age estimated from pink teeth is probably underestimated, but a more accurate age estimate can be obtained after adequate washing.

RATE OF ASPARTIC ACID RACEMIZATION IN BONE

There are no reports on rates of amino acid racemization in bones. To investigate the possibility of estimating age by evaluating amino acid residue racemization in human bones, a heating experiment was performed and the rate of aspartic acid racemization was determined using the Arrhenius equation. Assuming an annual mean temperature of 15 degrees C, the rate constant (k) for aspartic acid racemization in bone was calculated, and the racemization rate at 15 degrees C k (y) was 4.1036 x 10(-9)--much lower than that of dentin. These results suggest that it is more difficult to accurately determine age by analyzing aspartic acid residues in bone than in dentin.

Estimation of Age from Teeth by Amino Acid Racemization: Influence of Fixative

To determine the age of a subject from teeth accurately utilizing the racemization rates of amino acids, standard samples of the same tooth species from the same jaw are necessary as controls, as well as data for identification. However, standard teeth are generally stored in fixatives such as ethanol and formalin. We investigated and compared the degree of progression of racemization of dentinal aspartic acid in teeth stored in 95% ethanol, 10% formalin, or 10% neutral formalin fixatives. The racemization rate of dentinal aspartic acid in teeth stored in 10% neutral formalin was the highest, followed by that for teeth stored in 10% formalin then that for teeth stored in 95% ethanol. Teeth stored in these fixatives at 15 degrees C showed almost no progression of racemization. The racemization ratio (D/L ratio) in teeth extracted 10 years previously was almost unchanged from that at the time of extraction, and allowed an accurate evaluation of the subjects age at tooth extraction.

Age-related changes in the d-aspartic acid content of the teeth of the senescence-accelerated mouse

It is known that D-aspartic acid increases with age in dentine. Here, age-related changes in the D to L-aspartic acid (D/L) ratios of the lower teeth of two different sublines of the senescence-accelerated mouse (SAM), SAMP2/Iw (SAM, prone 2/Iwate) and SAMR1/Iw (SAM, resistant 1/Iwate) were measured by gas chromatography. The D/L ratio of the molars increased with advancing age, whereas that of the incisors did not. In mice younger than 6 months of age the D/L ratio of the molars from SAMP2/Iw tended to be higher than that from SAMR1/Iw, whereas the converse applied to older mice. Racemization in the molars occurred significantly faster in SAMR1/Iw than SAMP2/Iw (p = 0.01-0.001). Analysis according to the kind of tooth showed that the D/L ratio increased gradually in the order incisors < third molars < second molars < first molars, indicating that the ratio was higher the earlier the molars formed. As racemization depends upon the environmental temperature, the rectal temperatures of the mice were also examined. The rectal temperature of SAMP2/Iw was highest when they were 2 months old, but declined rapidly thereafter, whereas the rectal temperature of SAMRI/Iw was highest when they were 6 months old, after which it declined gradually. These results indicate that the D-aspartic acid contents of the molars of SAMR1/Iw and SAMP2/Iw increase with age in a different fashion and suggest that the fashion was determined by the body temperature, but not by the senescence-accelerated age.