- Email: [email protected]
Age estimation based on chemical approaches
Chapter 14
Age estimation based on chemical approaches Sara C. Zapico1,2, Cassandra M. DeGaglia2 and Joe Adserias-Garriga3 1
International Forensic Research Institute, Florida International University, Miami, FL, United States, 2Department of Anthropology, Smithsonian Institution, National Museum of Natural History, Washington, DC, United States, 3Forensic Anthropology Center, Texas State University, San Marcos, TX, United States
14.1 Introduction As described in the introductory chapter “The Evolution of Methodology in Biochemical Age Estimation,” chemical approaches for age-at-death estimation are based on chemical modifications during the aging process. This includes a broad range of processes, although all of them lead to produce changes on protein configuration. Racemization converts the natural form of L-aspartic acid on its specular form D-aspartic acid; lead accumulation seems to be more dependent on the environment; collagen cross-links refer to the stabilization of collagenous matrices; chemical composition of teeth is related to its change over time; advanced glycation end products (AGEs) refer to the reaction between reducing sugars and amino groups on proteins, leading to browning, fluorescence, crosslinking of proteins. Among all of these modifications, aspartic acid racemization is not only the most studied one but also it is the most precise and accurate technique, although it is not exempt of drawbacks. Current efforts try to improve this technique and explore the possibility of using other chemical methodologies with the same accuracy and precision.
14.2 Aspartic acid racemization L-Amino acids are commonly found in living systems as a result of the stereochemical specificity of enzymes which utilizes only the L-enantiomers [1]. Racemization is a natural process, which converts L-amino acids into their specular form, D-amino acids, leading to a racemic mixture. This reaction takes places in any metabolically stable protein that is not turned over Age Estimation. DOI: https://doi.org/10.1016/B978-0-12-814491-6.00014-5 Copyright © 2019 Elsevier Inc. All rights reserved.
199
200
SECTION | IV Biochemical approach of age estimation
during the lifetime of long-lived mammals. As a consequence of this process, these proteins will have alterations in their conformations leading to changes in their biological activities or chemical properties [2]. These alterations may contribute to the progressive changes associated with the aging process [3,4]. Although racemization occurs in all amino acids, it is well known that aspartic acid has one of the fastest racemization rates, making it suitable for forensic studies. The first study that demonstrated the applicability of this technique to forensic science was in 1975 when Helfman and Bada [1] analyzed the aspartic acid racemization in tooth enamel from living humans and found an increase in D/L ratios of aspartic acid with age. They confirmed these results using two techniques, gas chromatography (GC) and an aminoacid analyzer to separate diastereomeric dipeptides. Although they found this correlation in tooth enamel, they did not find it in human hemoglobin due to the rapid turnover of this protein. Since the 1975 study, this technique has been applied in a variety of tissues containing metabolically stable proteins, such as highly bradytrophic tissues, with a low rate of protein turnover [5], like dentin [6 8], cementum [9,10], human lens [2,11,12], human sclera [13], white matter of brain [14], intervertebral discs [5], elastin [15], bone [16 19], skin [20], epiglottis [21], and rib cartilage [22]. These studies found a positive correlation between aspartic acid racemization and age, although they also found variations. The tissue elected for analysis depends on circumstances. Although osteocalcin in cranial bone seems to be the best target for the estimation of age [23,24], the majority of studies point to dentin as the most accurate for age estimation in adults. Dentin methods yield a standard estimation error of 6 1.5 4 years and a correlation coefficient of 0.97 0.99 [13,25]. This was widely confirmed by different groups [26], in fact, Helfman and Bada [6] indicated that dentin is more reliable because it is in greater proportion than enamel and suffers less contamination and alteration such as attrition. In addition, the methodology applied for processing and analyzing racemization in human dentin is simple and it does not require much time since GC is the preferred method for enantiomers detection. However, this methodology has some drawbacks to overcome. It depends on the type of tooth because the period of dentin synthesis in the first years of life varies from tooth to tooth [8,27 30]. In addition, different values were observed when labial and lingual portions of the same tooth were compared [31]. A recent study from Sakuma et al. [32] compared the accuracy of using whole-tooth versus dentin samples, finding higher racemization rates when using the whole-tooth as well as a lower correlation coefficient and a higher standard deviation. Thus, the analysis of the “entire dentin of central longitudinal sections” and the standardization of sampling are recommended [30,33]. Another important disadvantage is the required use of healthy teeth as studies have noted the influence of caries on aspartic acid racemization
Age estimation based on chemical approaches Chapter | 14
201
estimates [34,35]. Also, as a first-order chemical reaction, racemization is influenced by temperature, making this technique unsuitable for corpses that have been exposed to higher temperatures [8,31,36]. Racemization can also be affected by contaminating proteins as is demonstrated in some cases of “pink teeth” [37,38]. One of the most serious drawbacks of this method is that several control teeth of the same type as the specimen to be estimated are required [39]. Ohtani et al. tried to solve this problem using standard specimens and artificial mixtures of D- and L-aspartic acids, which were prepared on the basis of the racemization ratios in central and lateral incisors. They demonstrated the usefulness of these artificial standards toward the applicability of this methodology [40]. Despite the drawbacks described above, this method is extremely consistent in comparison with conventional age estimation methods. These accurate estimates have the potential to be used in bodies with long postmortem intervals [39,41]. Currently, the applicability of aspartic acid racemization to forensic casework is limited, since only few institutes have the expertise to perform these analyses [13]. Aspartic acid racemization has helped identify unknown corpses in various criminal cases in Japan [39,42,43] and is also regularly performed at the Institute of Forensic Medicine in Du¨sseldorf for cases of unidentified corpses in North Rhine-Westphalia [13].
14.3 Lead accumulation Lead is one of the most significant pollutants in the environment [44], and its concentration on blood reflects exposure. In teeth, its concentration is a cumulative function of earlier exposure [45]. Dentin is the main site for lead deposition, providing evidence of early exposure until the tooth is extracted. In many countries, children’s teeth have been used as indicators of lead pollution [46,47], although the age range in these studies was narrow. In adults this range is very large, more than 50 years. If age affects lead accumulation, the relationship between duration of exposure and lead accumulation could be different between age groups [45,48,49]. Studies analyzing lead levels have been developed with health purposes, taking into account age and sex; thus, these studies can potentially be applied to forensic age estimation. Although some authors found a correlation of lead levels with age [45,50,51], others did not find any correlation [52]. Also, there is not an agreement on studies of children, some authors found a linear increase in lead’s concentration with age in deciduous teeth [53], while others only found an increase in one type of tooth [54] or negative correlation [55], and others did not find any correlations [56,57]. In adult teeth some authors found a correlation with age [58]. Other authors analyzed permanent teeth from children and adults finding a positive correlation between
202
SECTION | IV Biochemical approach of age estimation
lead accumulation and age [44,45,48,49,59]. The preferred technique of analyzing lead accumulation in teeth is atomic absorption spectrophotometry. Among these studies, only one has analyzed lead accumulation for forensic purposes [44]. The authors found a significant correlation between age and dentin lead levels in a nonoccupationally exposed Kuwaiti population with higher levels of dentin lead in males compared with females. The difference between estimated and real age using the regression formula was 1.3 1 4.8 years. Based on the scarce studies of lead accumulation and age, further research is needed to ensure the applicability of this technique to forensic age estimation.
14.4 Collagen cross-links The collagenous matrices of cartilage, bone, dentin, and other skeletal connective tissues are stabilized by covalent cross-links between collagen molecules [60,61]. The covalent cross-links are formed through the intermolecular reactions of aldehyde residues made on the protein monomers of lysyl oxidase [62]. Two cross-link pathways can be defined, one based on the precursor lysine aldehydes, the other on precursor hydroxylysine aldehydes [63]. Bailey and Shimokomaki [64] demonstrated a decrease in the reducible cross-links with increased age in samples of skin, tendon, articular cartilage, and bone of rat, bovine, and human. At the same time, they found an increase on two unidentified compounds. They demonstrated that the aldehyde-derived cross-links changed with age and these reducible bonds observed in young tissues are probably intermediate cross-links. The main maturation product in the hydroxylysine aldehyde pathway is pyridinoline. Moriguchi and Fujimoto [65] analyzed this pathway in rat and human costal cartilage and Achilles tendon. They found an increase in pyridinoline content with age until the time of physiological maturity (about 20 years of age in humans). The pyridinoline content began to decrease after about 30 years of age in humans, suggesting its transformation into other compounds. Later, the concentration of borohydride-reducible and mature hydroxypyridinium cross-linking amino acids was measured in collagen samples of bone and cartilage from human subjects aged from 1 month to 80 years [66]. A decrease in the content of the total reducible cross-links from birth until about 25 years of age was found. However, the content of the mature cross-links shows an increasing trend from birth to 25 years. After that, the contents of both types of cross-link level off, with a down tendency still evident for the reducible compounds. Other groups analyzed collagen in dentin. Walters and Eyre [67] found an increase of hydroxypyridinium residues in bovine and human dentin with age, as the content of reducible cross-links fell. However, a significant level of reducible cross-links remains throughout the tooth’s adult life. The
Age estimation based on chemical approaches Chapter | 14
203
techniques applied to analyzed cross-links were chromatography and fluorescence detection. There is only one cross-link study developed for forensic purposes [68]. Martin-de las Heras et al. analyzed another component of nonreducible cross-links, deoxypyridinoline (DPD) in permanent molars from patients between 15 and 73 years old. They performed different extractions of dentin proteins and quantified the DPD levels by an enzyme immunoassay. Although they found an increase in the DPD ratio with age, the error of this technique was high ( 1 14.9 years) at 65% level of confidence.
14.5 Chemical composition of teeth With age, teeth become weaker and more prone to fracture. Due to the process of aging, a noncarious transparent dentin is formed, starting at the apex of the root and sometimes extending into the coronal dentin [69 71]. The transparency of teeth is due to the mineralization of the dentin substance around the dentinal tubules (peritubular dentin) and the gradual reduction of the dentinal tubules [72]. Scanning electron microscopic studies were developed by Kosa et al. in order to analyze the finer structure of dentin associated with age. According to these studies, dentinal tubules become thinner with increasing age, although it is not significant. Also associated with aging is the widening of the intertubular area and aggregation of the peritubular matrix. All of these changes are accompanied by changes in chemical composition [73]. In 1989, Kosa et al. studied the composition of the bones using electron probe microanalysis [74], finding a significant decrease in the Ca:P ratio in older individuals. In dentin [73], they found a decrease in the Ca:P ratio with age in peritubular (hypermineralized) dentin. Also, the weight of phosphorus is more closely correlated with age, but not the amount of Ca. Raman spectroscopy is a useful tool to study the chemical composition of teeth. Tramini et al. [75] used Raman microspectrometry to analyze the dentinal part of the tooth. Four different dentinal areas were defined on each tooth, coronal dentin, dentin cementum limit, root dentin, and apical dentin. Finally, stretch bands were identified, generating a multivariate analysis model. The partial correlation coefficient between predictors and age provided mainly a high value with a mean error of 5 years. Also, the authors found slight differences between males and females. Despite these results, some predictors in the model seemed to follow a nonlinear progression with age. Raman spectroscopy coupled with ultraviolet resonance Raman spectroscopy (UVRRS) has been used by Ager et al. [76] to analyze age-induced changes in cortical bone, finding alterations in the amide I band. By applying similar techniques in human teeth [77], they also found an increase in the
204
SECTION | IV Biochemical approach of age estimation
amide I peak height in dehydrated and demineralized dentin, as a result of the increased interaction between collagen fibrils caused by stretching and intrafibrillar movement.
14.6 Advanced glycation end products The Maillard reaction is a complex series of reactions between reducing sugars and amino groups of proteins, which leads to browning, fluorescence, and cross-linking of protein [78]. AGEs, formed during the later stages of the Maillard reaction, accumulate in long-lived tissue proteins and may contribute to the development of complications in aging [79]. Human aging is associated with a stiffening of tissues that are rich in extracellular matrix and long-lived proteins, such as skeletal muscle, tendons, joints, bone, heart, arteries, lung, skin, and lens [80]. Glyoxal (GO), methylglyoxal (MGO), and deoxyglucosones belong to a series of dicarbonyl compounds, identified as intermediates in the Maillard reaction. GO and MGO react with lysine and arginine residues in protein to yield well-characterized compounds, such as the N-(carbosyalkyl)lysines, N-(carboxymethyl)lysine (CML) and N-(1-(1-carbooxy)ethyl) lysine (CEL), and imidazolones and dehydroimidazolones [81 83]. The majority of the studies related to AGEs were developed with aging purposes. They found an accumulation of AGEs in permanent and long-lived proteins during an individual’s lifetime, mainly focusing on pentosidine, CML, CEL, and/or furosine in dentin [84], crystalline lens [85], articular cartilage [86,87], and skin collagen [88]. A study conducted by Sato et al. [89] described an increase of AGEs with age in hippocampal pyramidal neurons using an antibody against AGEs to demonstrate an immunohistochemical. Although they did not find an AGE signal in young individuals, the signal increases with age, accumulating higher amounts in older individuals. The authors suggested the possibility of forensic uses of this technique in fire death cases since they found this correlation with age. Pilin et al. [90 92] demonstrated the age-related color changes in intervertebral disc excisions, Achilles tendons, and rib cartilage as a result of accumulation of AGEs. However, the color changes and correlation coefficients were different among tissues, being more conspicuous in the rib cartilage than in the intervertebral discs, and finding almost no relation with age in Achilles tendon. In addition, age estimation was only reliable up to 45 years old, beyond that the method is less accurate. The color changes caused by aging and the utilization of this effect for age estimation were studied on hard dental tissues. Some authors demonstrated that teeth become more yellow with age [93,94]. Using a spectroradiometry technique to measure these color changes, Martin-de las Heras et al. [95] tried to correlate these changes with age. Although they found a correlation, the error averaged 13.7 years. They also found differences in
Age estimation based on chemical approaches Chapter | 14
205
dental color depending on the postmortem interval. Thus, this technique is not suitable for samples with extended postmortem intervals. In a recent study, Greis et al. [96] analyzed AGEs in dentin for their applicability in forensic age estimation. They used high performance liquid chromatography (HPLC) to determine the levels of pentosidine and found a high correlation, although there was an error of 1 9.4 years. Unfortunately, this technique cannot be applied in teeth of individuals with diabetes mellitus, because AGEs are accumulated in diabetes. The same study indicated a lack of suitability for use in fire death cases, although they posited the possibility of the use of this technique in combination with aspartic acid racemization.
14.7 Discussion Among the chemical techniques presented here, aspartic acid racemization seems to be the best to apply to determine age in forensic cases. It can be used in different tissues with high accuracy, although the preferable tissue is dentin [32,39]. In addition, the technique GC is available for forensic labs, although currently only two labs apply this technique to forensic casework [13]. Despite the success of aspartic acid racemization, it is not exempt from disadvantages. It cannot be used in bodies exposed to high temperatures because the reaction is affected by temperature [31]. Also, several control teeth are needed [32,33,40], although it is possible to overcome this drawback using an artificial mixture that could be continuously available in forensic laboratories [40]. The majority of lead accumulation studies were developed for health purposes, and these studies are not in agreement with respect to the accumulation of lead with age. The only study developed in a forensic context [44] found a significant correlation between age and dentin lead levels. However, this is only applicable to Kuwaiti populations because lead levels change between geographic regions as these levels depend on industrial activity and environmental conditions. Thus, population-specific references are required to develop a formula for the estimation of age-at-death with this technique. A decrease in the reducible collagen crosslinks associated with increased age was demonstrated in different studies and tissues [67], although these studies were developed for better understanding of the aging phenomena. The only study carried out with a forensic focus [68] did find a correlation with age in dentin. However, the error on age estimation was high and the technique is time consuming. The relationship between changes in the chemical composition of teeth with age using different methodologies is less developed. The latest studies point to Raman spectroscopy as an elected technique [97]. However, it requires a wide knowledge of chemistry to correctly relate the results of dental composition with age.
206
SECTION | IV Biochemical approach of age estimation
AGEs are widely studied with aging and age-related disease purposes [78]. Forensic studies point to a correlation of AGEs with age, such as in hippocampal pyramidal neurons [89]. Other studies are based on color changes due to AGEs [90 92]; however, these studies indicate differences in the correlation between tissues. A correlation based on color was found in dentin, but the error rate was high [95]. A recent study [96] analyzed levels of pentosidine in dentin finding a high correlation with age, although the error between estimated and real age was high. In addition, this technique is not suitable for teeth from individuals with diabetes and/or in fire death cases, although this study pointed out the possibility of using this technique in conjunction with aspartic acid racemization.
References [1] Helfman PM, Bada JL. Aspartic acid racemization in tooth enamel from living humans. Proc Natl Acad Sci USA 1975;72(8):2891 4. [2] Masters PM, Bada JL, Zigler Jr. JS. Aspartic acid racemisation in the human lens during ageing and in cataract formation. Nature 1977;268(5615):71 3. [3] Helfman PM, Bada JL, Shou MY. Considerations on the role of aspartic acid racemization in the aging process. Gerontology 1977;23(6):419 25. [4] Kuhn W. Possible relation between optical activity and aging. Adv Enzymol Related Subj Biochem 1958;20:1 29. [5] Ritz S, Schutz HW. Aspartic acid racemization in intervertebral discs as an aid to postmortem estimation of age at death. J Forensic Sci 1993;38(3):633 40. [6] Helfman PM, Bada JL. Aspartic acid racemisation in dentine as a measure of ageing. Nature 1976;262(5566):279 81. [7] Ohtani S. Estimation of age from dentin by using the racemization reaction of aspartic acid. Am J Forensic Med Pathol 1995;16(2):158 61. [8] Ritz S, Schutz HW, Schwarzer B. The extent of aspartic acid racemization in dentin: a possible method for a more accurate determination of age at death? Zeitschrift fur Rechtsmedizin. J Leg Med 1990;103(6):457 62. [9] Ohtani S, Sugimoto H, Sugeno H, Yamamoto S, Yamamoto K. Racemization of aspartic acid in human cementum with age. Arch Oral Biol 1995;40(2):91 5. [10] Ohtani S. Studies on age estimation using racemization of aspartic acid in cementum. J Forensic Sci 1995;40(5):805 7. [11] Garner WH, Spector A. Racemization in human lens: evidence of rapid insolubilization of specific polypeptides in cataract formation. Proc Natl Acad Sci USA 1978;75 (8):3618 20. [12] Masters PM, Bada JL, Zigler Jr. JS. Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts. Proc Natl Acad Sci USA 1978;75(3):1204 8. [13] Klumb K, Matzenauer C, Reckert A, Lehmann K, Ritz-Timme S. Age estimation based on aspartic acid racemization in human sclera. Int J Leg Med 2016;130(1):207 11. Available from: https://doi.org/10.1007/s00414-015-1255-6. [14] Man EH, Sandhouse ME, Burg J, Fisher GH. Accumulation of D-aspartic acid with age in the human brain. Science 1983;220(4604):1407 8.
Age estimation based on chemical approaches Chapter | 14
207
[15] Dobberstein RC, Tung SM, Ritz-Timme S. Aspartic acid racemisation in purified elastin from arteries as basis for age estimation. Int J Leg Med 2010;124(4):269 75. Available from: https://doi.org/10.1007/s00414-009-0392-1. [16] Ohtani S, Matsushima Y, Kobayashi Y, Kishi K. Evaluation of aspartic acid racemization ratios in the human femur for age estimation. J Forensic Sci 1998;43(5):949 53. [17] Ohtani S, Yamamoto T, Matsushima Y, Kobayashi Y. Changes in the amount of Daspartic acid in the human femur with age. Growth Dev Aging: GDA 1998;62 (4):141 8. [18] Ohtani S. Rate of aspartic acid racemization in bone. Am J Forensic Med Pathol 1998;19 (3):284 7. [19] Ohtani S. Technical notes for age estimation using the femur: influence of various analytical conditions on D-aspartic acid contents. Int J Leg Med 2002;116(6):361 4. [20] Tiplamaz S, Goren MZ, Yurtsever NT. Estimation of chronological age from postmortem tissues based on amino acid racemization. J Forensic Sci 2018. Available from: https:// doi.org/10.1111/1556-4029.13737. [21] Matzenauer C, Reckert A, Ritz-Timme S. Estimation of age at death based on aspartic acid racemization in elastic cartilage of the epiglottis. Int J Leg Med 2014;128 (6):995 1000. Available from: https://doi.org/10.1007/s00414-013-0940-6. [22] Ohtani S, Matsushima Y, Kobayashi Y, Yamamoto T. 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. J Forensic Sci 2002;47(1):32 6. [23] Ritz S, Turzynski A, Schutz HW. Estimation of age at death based on aspartic acid racemization in noncollagenous bone proteins. Forensic Sci Int 1994;69(2):149 59. [24] Ritz S, Turzynski A, Schutz HW, Hollmann A, Rochholz G. Identification of osteocalcin as a permanent aging constituent of the bone matrix: basis for an accurate age at death determination. Forensic Sci Int 1996;77(1-2):13 26. [25] Ritz-Timme S, Cattaneo C, Collins MJ, Waite ER, Schutz HW, Kaatsch HJ, et al. Age estimation: the state of the art in relation to the specific demands of forensic practise. Int J Leg Med 2000;113(3):129 36. [26] Yekkala R, Meers C, Van Schepdael A, Hoogmartens J, Lambrichts I, Willems G. Racemization of aspartic acid from human dentin in the estimation of chronological age. Forensic Sci Int 2006;159(Suppl 1):S89 94. Available from: https://doi.org/10.1016/j. forsciint.2006.02.022. [27] Ohtani S, Yamamoto T. 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. J Forensic Sci 2005;50(5):1020 7. [28] Yekkala R, Meers C, Hoogmartens J, Lambrichts I, Willems G, Van Schepdael A. An improved sample preparation for an LC method used in the age estimation based on aspartic acid racemization from human dentin. J Sep Sci 2007;30(1):118 21. [29] Ohtani S, Yamamoto K. Estimation of ages from racemization of an amino acid in teeth—assessment of errors under various experimental conditions. Nihon hoigaku zasshi 1991;45(2):124 7. [30] Ohtani S, Yamamoto K. Age estimation using the racemization of amino acid in human dentin. J Forensic Sci 1991;36(3):792 800. [31] Ritz S, Schutz HW, Peper C. Postmortem estimation of age at death based on aspartic acid racemization in dentin: its applicability for root dentin. Int J Leg Med 1993;105 (5):289 93.
208
SECTION | IV Biochemical approach of age estimation
[32] Sakuma A, Ohtani S, Saitoh H, Iwase H. Comparative analysis of aspartic acid racemization methods using whole-tooth and dentin samples. Forensic Sci Int 2012;223(13):198 201. Available from: https://doi.org/10.1016/j.forsciint.2012.08.043. [33] Ohtani S, Yamamoto K. Estimation of age from a tooth by means of racemization of an amino acid, especially aspartic acid—comparison of enamel and dentin. J Forensic Sci 1992;37(4):1061 7. [34] Griffin RC, Moody H, Penkman KE, Collins MJ. The application of amino acid racemization in the acid soluble fraction of enamel to the estimation of the age of human teeth. Forensic Sci Int 2008;175(1):11 16. Available from: https://doi.org/10.1016/j. forsciint.2007.04.226. [35] Sirin N, Matzenauer C, Reckert A, Ritz-Timme S. Age estimation based on aspartic acid racemization in dentine: what about caries-affected teeth? Int J Leg Med 2018;132 (2):623 8. Available from: https://doi.org/10.1007/s00414-017-1667-6. [36] Ohtani S. Different racemization ratios in dentin from different locations within a tooth. Growth Dev Aging: GDA 1997;61(2):93 9. [37] Ohtani S, Yamada Y, Yamamoto I. Improvement of age estimation using amino acid racemization in a case of pink teeth. Am J Forensic Med Pathol 1998;19(1):77 9. [38] Sakuma A, Saitoh H, Ishii N, Iwase H. The effects of racemization rate for age estimation of pink teeth. J Forensic Sci 2015;60(2):450 2. Available from: https://doi.org/10.1111/ 1556-4029.12653. [39] Ohtani S, Yamamoto T. Age estimation by amino acid racemization in human teeth. J Forensic Sci 2010;55(6):1630 3. Available from: https://doi.org/10.1111/j.15564029.2010.01472.x. [40] Ohtani S, Abe I, Yamamoto T. An application of D- and L-aspartic acid mixtures as standard specimens for the chronological age estimation. J Forensic Sci 2005;50 (6):1298 302. [41] Ritz-Timme S, Rochholz G, Schutz HW, Collins MJ, Waite ER, Cattaneo C, et al. Quality assurance in age estimation based on aspartic acid racemisation. International J Leg Med 2000;114(1-2):83 6. [42] Ohtani S, Yamagishi M, Ogasawara M, Yamamoto K. Age estimation of two unidentified bodies by amino acid racemization in their teeth. Bull Kanagawa Dent Coll: BKDC/KDS 1990;18(1):23 7. [43] Ohtani S, Utsunomiya J, Minoshima T, Yamamoto K. Tooth-based age estimation of an adipocerated cadaver using the amino acid racemization method. Nihon hoigaku zasshi 1994;48(4):279 81. [44] Al-Qattan SI, Elfawal MA. Significance of teeth lead accumulation in age estimation. J Forensic Legal Med 2010;17(6):325 8. Available from: https://doi.org/10.1016/j. jflm.2010.05.001. [45] Steenhout A, Pourtois M. Lead accumulation in teeth as a function of age with different exposures. Br J Ind Med 1981;38(3):297 303. [46] Fosse G, Justesen NP. Lead in deciduous teeth of Norwegian children. Arch Environ Health 1978;33(4):166 75. [47] Haavikko K, Anttila A, Helle A, Vuori E. Lead concentrations of enamel and dentine of deciduous teeth of children from two Finnish towns. Arch Environ Health 1984;39 (2):78 84. [48] Bercovitz K, Laufer D. Lead accumulation in teeth of patients suffering from gastrointestinal ulcers. Sci Total Environ 1991;101(3):229 34.
Age estimation based on chemical approaches Chapter | 14
209
[49] Bercovitz K, Laufer D. Age and gender influence on lead accumulation in root dentine of human permanent teeth. Arch Oral Biol 1991;36(9):671 3. [50] Drasch GA, Ott J. Lead in human bones. Investigations on an occupationally non-exposed population in southern Bavaria (F.R.G.). II. Children. Sci Total Environ 1988;68:61 9. [51] Somervaille LJ, Chettle DR, Scott MC. In vivo measurement of lead in bone using x-ray fluorescence. Phys Med Biol 1985;30(9):929 43. [52] Nusbaum RE, Butt EM, Gilmour TC, Didio SL. Relation of air pollutants to trace metals in bone. Arch Environ Health 1965;10:227 32. [53] Altshuller LF, Halak DB, Landing BH, Kehoe RA. Deciduous teeth as an index of body burden of lead. J Pediatr 1962;60:224 9. [54] Mackie AC, Stephens R, Townshend A. Tooth lead levels in Birmingham children. Arch Environ Health 1977;32(4):178 85. [55] Pinchin MJ, Newham J, Thompson RP. Lead, copper, and cadmium in teeth of normal and mentally retarded children. Clin Chim Acta 1978;85(1):89 94. [56] Habercam JW, Keil JE, Reigart JR, Croft HW. Lead content of human blood, hair, and deciduous teeth: correlation with environmental factors and growth. J Dent Res 1974;53 (5):1160 3. [57] Holtzman RB, Lucas Jr. HF, Ilcewicz FH. The concentration of lead in human bone. ANL-7615. ANL Rep 1968;43 9. [58] Strehlow CD, Kneip TJ. The distribution of lead and zinc in the human skeleton. Am Ind Hyg Assoc J 1969;30(4):372 8. Available from: https://doi.org/10.1080/00028896909343140. [59] Bercovitz K, Laufer D. Lead levels in teeth of long date and new immigrants in several cities in Israel—preliminary results. Public Health Rev 1991;19(1-4):141 6. [60] Eyre D. Collagen cross-linking amino acids. Methods Enzymol 1987;144:115 39. [61] Mechanic G, Gallop PM, Tanzer ML. The nature of crosslinking in collagens from mineralized tissues. Biochem Biophys Res Commun 1971;45(3):644 53. [62] Piez KA. Cross-linking of collagen and elastin. Annu Rev Biochem 1968;37:547 70. Available from: https://doi.org/10.1146/annurev.bi.37.070168.002555. [63] Eyre DR, Paz MA, Gallop PM. Cross-linking in collagen and elastin. Annu Rev Biochem 1984;53:717 48. Available from: https://doi.org/10.1146/annurev.bi.53.070184.003441. [64] Bailey AJ, Shimokomaki MS. Age related changes in the reducible cross-links of collagen. FEBS Lett 1971;16(2):86 8. [65] Moriguchi T, Fujimoto D. Age-related changes in the content of the collagen crosslink, pyridinoline. J Biochem 1978;84(4):933 5. [66] Eyre DR, Dickson IR, Van Ness K. Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues. Biochem J 1988;252(2):495 500. [67] Walters C, Eyre DR. Collagen crosslinks in human dentin: increasing content of hydroxypyridinium residues with age. Calcif Tissue Int 1983;35(4-5):401 5. [68] Martin-de las Heras S, Valenzuela A, Villanueva E. Deoxypyridinoline crosslinks in human dentin and estimation of age. Int J Leg Med 1999;112(4):222 6. [69] Micheletti Cremasco M. Dental histology: study of aging processes in root dentine. Bollettino della Societa italiana di biologia sperimentale 1998;74(3-4):19 28. [70] Vasiliadis L, Darling AI, Levers BG. The histology of sclerotic human root dentine. Arch Oral Biol 1983;28(8):693 700. [71] Vasiliadis L, Darling AI, Levers BG. The amount and distribution of sclerotic human root dentine. Arch Oral Biol 1983;28(7):645 9.
210
SECTION | IV Biochemical approach of age estimation
[72] Amprino R, Engstrom A. Studies on x ray absorption and diffraction of bone tissue. Acta Anat 1952;15(1-2):1 22. [73] Kosa F, Antal A, Farkas I. Electron probe microanalysis of human teeth for the determination of individual age. Med Sci Law 1990;30(2):109 14. [74] Kosa F, Farkas I, Wittman G. [Microprobe study of bones in the determination of individual age]. Morphologiai es igazsagugyi orvosi szemle 1989;29(3):227 32. [75] Tramini P, Bonnet B, Sabatier R, Maury L. A method of age estimation using Raman microspectrometry imaging of the human dentin. Forensic Sci Int 2001;118(1):1 9. [76] Ager JW, Nalla RK, Breeden KL, Ritchie RO. Deep-ultraviolet Raman spectroscopy study of the effect of aging on human cortical bone. J Biomed Opt 2005;10(3):034012. Available from: https://doi.org/10.1117/1.1924668. [77] Ager 3rd JW, Nalla RK, Balooch G, Kim G, Pugach M, Habelitz S, et al. On the increasing fragility of human teeth with age: a deep-UV resonance Raman study. J Bone Miner Res 2006;21(12):1879 87. Available from: https://doi.org/10.1359/jbmr.060816. [78] Baynes JW. The role of AGEs in aging: causation or correlation. Exp Gerontol 2001;36 (9):1527 37. [79] Thorpe SR, Baynes JW. Role of the Maillard reaction in diabetes mellitus and diseases of aging. Drugs Aging 1996;9(2):69 77. [80] Monnier VM, Mustata GT, Biemel KL, Reihl O, Lederer MO, Zhenyu D, et al. Crosslinking of the extracellular matrix by the maillard reaction in aging and diabetes: an update on “a puzzle nearing resolution”. Ann N Y Acad Sci 2005;1043:533 44. Available from: https://doi.org/10.1196/annals.1333.061. [81] Ahmed MU, Brinkmann Frye E, Degenhardt TP, Thorpe SR, Baynes JW. N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem J 1997;324(Pt 2):565 70. [82] Ahmed MU, Thorpe SR, Baynes JW. Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein. J Biol Chem 1986;261 (11):4889 94. [83] Henle T, Walter AW, Klostermeyer H. A simple method for the preparation of furosine and pyridosine reference material. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung 1994;198(1):66 7. [84] Miura J, Nishikawa K, Kubo M, Fukushima S, Hashimoto M, Takeshige F, et al. Accumulation of advanced glycation end-products in human dentine. Arch Oral Biol 2014;59(2):119 24. Available from: https://doi.org/10.1016/j.archoralbio.2013.10.012. [85] Marques C, Ramalho JS, Pereira P, Mota MC. Bendazac decreases in vitro glycation of human lens crystallins. Decrease of in vitro protein glycation by bendazac. Doc Ophthalmol 1995;90(4):395 404. [86] Verzijl N, DeGroot J, Ben ZC, Brau-Benjamin O, Maroudas A, Bank RA, et al. Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: a possible mechanism through which age is a risk factor for osteoarthritis. Arthritis Rheum 2002;46(1):114 23 doi:10.1002/1529-0131 (200201)46:1 , 114::AID-ART10025 . 3.0.CO;2-P. [87] Verzijl N, DeGroot J, Oldehinkel E, Bank RA, Thorpe SR, Baynes JW, et al. Age-related accumulation of Maillard reaction products in human articular cartilage collagen. Biochem J 2000;350(Pt 2):381 7. [88] Dyer DG, Dunn JA, Thorpe SR, Bailie KE, Lyons TJ, McCance DR, et al. Accumulation of Maillard reaction products in skin collagen in diabetes and aging. J Clin Invest 1993;91 (6):2463 9. Available from: https://doi.org/10.1172/JCI116481.
Age estimation based on chemical approaches Chapter | 14
211
[89] Sato Y, Kondo T, Ohshima T. Estimation of age of human cadavers by immunohistochemical assessment of advanced glycation end products in the hippocampus. Histopathology 2001;38(3):217 20. [90] Pilin A, Pudil F, Bencko V. The image analysis of colour changes of different human tissues in the relation to the age. Part 2: practical applicability. Soudni lekarstvi 2007;52 (3):36 42. [91] Pilin A, Pudil F, Bencko V. The image analysis of colour changes of different human tissues in the relation to the age. Part 1. Methodological approach. Soudni lekarstvi / casopis Sekce soudniho lekarstvi Cs lekarske spolecnosti J Ev Purkyne 2007;52(2):26 30. [92] Pilin A, Pudil F, Bencko V. Changes in colour of different human tissues as a marker of age. Int J Leg Med 2007;121(2):158 62. Available from: https://doi.org/10.1007/s00414006-0136-4. [93] Ten Cate AR, Thompson GW, Dickinson JB, Hunter HA. The estimation of age of skeletal remains from the colour of roots of teeth. Dent J 1977;43(2):83 6. [94] Lackovic KP, Wood RE. Tooth root colour as a measure of chronological age. J Forensic Odonto-Stomatol 2000;18(2):37 45. [95] Martin-de las Heras S, Valenzuela A, Bellini R, Salas C, Rubino M, Garcia JA. Objective measurement of dental color for age estimation by spectroradiometry. Forensic Sci Int 2003;132(1):57 62. [96] Greis F, Reckert A, Fischer K, Ritz-Timme S. Analysis of advanced glycation end products (AGEs) in dentine: useful for age estimation? Int J Leg Med 2017. Available from: https://doi.org/10.1007/s00414-017-1671-x. [97] Heudt L, Debois D, Zimmerman TA, Kohler L, Bano F, Partouche F, et al. Raman spectroscopy and laser desorption mass spectrometry for minimal destructive forensic analysis of black and color inkjet printed documents. Forensic Sci Int 2012;219(1-3):64 75. Available from: https://doi.org/10.1016/j.forsciint.2011.12.001.
Age estimation based on chemical approaches Sara C. Zapico1,2, Cassandra M. DeGaglia2 and Joe Adserias-Garriga3 1
International Forensic Research Institute, Florida International University, Miami, FL, United States, 2Department of Anthropology, Smithsonian Institution, National Museum of Natural History, Washington, DC, United States, 3Forensic Anthropology Center, Texas State University, San Marcos, TX, United States
14.1 Introduction As described in the introductory chapter “The Evolution of Methodology in Biochemical Age Estimation,” chemical approaches for age-at-death estimation are based on chemical modifications during the aging process. This includes a broad range of processes, although all of them lead to produce changes on protein configuration. Racemization converts the natural form of L-aspartic acid on its specular form D-aspartic acid; lead accumulation seems to be more dependent on the environment; collagen cross-links refer to the stabilization of collagenous matrices; chemical composition of teeth is related to its change over time; advanced glycation end products (AGEs) refer to the reaction between reducing sugars and amino groups on proteins, leading to browning, fluorescence, crosslinking of proteins. Among all of these modifications, aspartic acid racemization is not only the most studied one but also it is the most precise and accurate technique, although it is not exempt of drawbacks. Current efforts try to improve this technique and explore the possibility of using other chemical methodologies with the same accuracy and precision.
14.2 Aspartic acid racemization L-Amino acids are commonly found in living systems as a result of the stereochemical specificity of enzymes which utilizes only the L-enantiomers [1]. Racemization is a natural process, which converts L-amino acids into their specular form, D-amino acids, leading to a racemic mixture. This reaction takes places in any metabolically stable protein that is not turned over Age Estimation. DOI: https://doi.org/10.1016/B978-0-12-814491-6.00014-5 Copyright © 2019 Elsevier Inc. All rights reserved.
199
200
SECTION | IV Biochemical approach of age estimation
during the lifetime of long-lived mammals. As a consequence of this process, these proteins will have alterations in their conformations leading to changes in their biological activities or chemical properties [2]. These alterations may contribute to the progressive changes associated with the aging process [3,4]. Although racemization occurs in all amino acids, it is well known that aspartic acid has one of the fastest racemization rates, making it suitable for forensic studies. The first study that demonstrated the applicability of this technique to forensic science was in 1975 when Helfman and Bada [1] analyzed the aspartic acid racemization in tooth enamel from living humans and found an increase in D/L ratios of aspartic acid with age. They confirmed these results using two techniques, gas chromatography (GC) and an aminoacid analyzer to separate diastereomeric dipeptides. Although they found this correlation in tooth enamel, they did not find it in human hemoglobin due to the rapid turnover of this protein. Since the 1975 study, this technique has been applied in a variety of tissues containing metabolically stable proteins, such as highly bradytrophic tissues, with a low rate of protein turnover [5], like dentin [6 8], cementum [9,10], human lens [2,11,12], human sclera [13], white matter of brain [14], intervertebral discs [5], elastin [15], bone [16 19], skin [20], epiglottis [21], and rib cartilage [22]. These studies found a positive correlation between aspartic acid racemization and age, although they also found variations. The tissue elected for analysis depends on circumstances. Although osteocalcin in cranial bone seems to be the best target for the estimation of age [23,24], the majority of studies point to dentin as the most accurate for age estimation in adults. Dentin methods yield a standard estimation error of 6 1.5 4 years and a correlation coefficient of 0.97 0.99 [13,25]. This was widely confirmed by different groups [26], in fact, Helfman and Bada [6] indicated that dentin is more reliable because it is in greater proportion than enamel and suffers less contamination and alteration such as attrition. In addition, the methodology applied for processing and analyzing racemization in human dentin is simple and it does not require much time since GC is the preferred method for enantiomers detection. However, this methodology has some drawbacks to overcome. It depends on the type of tooth because the period of dentin synthesis in the first years of life varies from tooth to tooth [8,27 30]. In addition, different values were observed when labial and lingual portions of the same tooth were compared [31]. A recent study from Sakuma et al. [32] compared the accuracy of using whole-tooth versus dentin samples, finding higher racemization rates when using the whole-tooth as well as a lower correlation coefficient and a higher standard deviation. Thus, the analysis of the “entire dentin of central longitudinal sections” and the standardization of sampling are recommended [30,33]. Another important disadvantage is the required use of healthy teeth as studies have noted the influence of caries on aspartic acid racemization
Age estimation based on chemical approaches Chapter | 14
201
estimates [34,35]. Also, as a first-order chemical reaction, racemization is influenced by temperature, making this technique unsuitable for corpses that have been exposed to higher temperatures [8,31,36]. Racemization can also be affected by contaminating proteins as is demonstrated in some cases of “pink teeth” [37,38]. One of the most serious drawbacks of this method is that several control teeth of the same type as the specimen to be estimated are required [39]. Ohtani et al. tried to solve this problem using standard specimens and artificial mixtures of D- and L-aspartic acids, which were prepared on the basis of the racemization ratios in central and lateral incisors. They demonstrated the usefulness of these artificial standards toward the applicability of this methodology [40]. Despite the drawbacks described above, this method is extremely consistent in comparison with conventional age estimation methods. These accurate estimates have the potential to be used in bodies with long postmortem intervals [39,41]. Currently, the applicability of aspartic acid racemization to forensic casework is limited, since only few institutes have the expertise to perform these analyses [13]. Aspartic acid racemization has helped identify unknown corpses in various criminal cases in Japan [39,42,43] and is also regularly performed at the Institute of Forensic Medicine in Du¨sseldorf for cases of unidentified corpses in North Rhine-Westphalia [13].
14.3 Lead accumulation Lead is one of the most significant pollutants in the environment [44], and its concentration on blood reflects exposure. In teeth, its concentration is a cumulative function of earlier exposure [45]. Dentin is the main site for lead deposition, providing evidence of early exposure until the tooth is extracted. In many countries, children’s teeth have been used as indicators of lead pollution [46,47], although the age range in these studies was narrow. In adults this range is very large, more than 50 years. If age affects lead accumulation, the relationship between duration of exposure and lead accumulation could be different between age groups [45,48,49]. Studies analyzing lead levels have been developed with health purposes, taking into account age and sex; thus, these studies can potentially be applied to forensic age estimation. Although some authors found a correlation of lead levels with age [45,50,51], others did not find any correlation [52]. Also, there is not an agreement on studies of children, some authors found a linear increase in lead’s concentration with age in deciduous teeth [53], while others only found an increase in one type of tooth [54] or negative correlation [55], and others did not find any correlations [56,57]. In adult teeth some authors found a correlation with age [58]. Other authors analyzed permanent teeth from children and adults finding a positive correlation between
202
SECTION | IV Biochemical approach of age estimation
lead accumulation and age [44,45,48,49,59]. The preferred technique of analyzing lead accumulation in teeth is atomic absorption spectrophotometry. Among these studies, only one has analyzed lead accumulation for forensic purposes [44]. The authors found a significant correlation between age and dentin lead levels in a nonoccupationally exposed Kuwaiti population with higher levels of dentin lead in males compared with females. The difference between estimated and real age using the regression formula was 1.3 1 4.8 years. Based on the scarce studies of lead accumulation and age, further research is needed to ensure the applicability of this technique to forensic age estimation.
14.4 Collagen cross-links The collagenous matrices of cartilage, bone, dentin, and other skeletal connective tissues are stabilized by covalent cross-links between collagen molecules [60,61]. The covalent cross-links are formed through the intermolecular reactions of aldehyde residues made on the protein monomers of lysyl oxidase [62]. Two cross-link pathways can be defined, one based on the precursor lysine aldehydes, the other on precursor hydroxylysine aldehydes [63]. Bailey and Shimokomaki [64] demonstrated a decrease in the reducible cross-links with increased age in samples of skin, tendon, articular cartilage, and bone of rat, bovine, and human. At the same time, they found an increase on two unidentified compounds. They demonstrated that the aldehyde-derived cross-links changed with age and these reducible bonds observed in young tissues are probably intermediate cross-links. The main maturation product in the hydroxylysine aldehyde pathway is pyridinoline. Moriguchi and Fujimoto [65] analyzed this pathway in rat and human costal cartilage and Achilles tendon. They found an increase in pyridinoline content with age until the time of physiological maturity (about 20 years of age in humans). The pyridinoline content began to decrease after about 30 years of age in humans, suggesting its transformation into other compounds. Later, the concentration of borohydride-reducible and mature hydroxypyridinium cross-linking amino acids was measured in collagen samples of bone and cartilage from human subjects aged from 1 month to 80 years [66]. A decrease in the content of the total reducible cross-links from birth until about 25 years of age was found. However, the content of the mature cross-links shows an increasing trend from birth to 25 years. After that, the contents of both types of cross-link level off, with a down tendency still evident for the reducible compounds. Other groups analyzed collagen in dentin. Walters and Eyre [67] found an increase of hydroxypyridinium residues in bovine and human dentin with age, as the content of reducible cross-links fell. However, a significant level of reducible cross-links remains throughout the tooth’s adult life. The
Age estimation based on chemical approaches Chapter | 14
203
techniques applied to analyzed cross-links were chromatography and fluorescence detection. There is only one cross-link study developed for forensic purposes [68]. Martin-de las Heras et al. analyzed another component of nonreducible cross-links, deoxypyridinoline (DPD) in permanent molars from patients between 15 and 73 years old. They performed different extractions of dentin proteins and quantified the DPD levels by an enzyme immunoassay. Although they found an increase in the DPD ratio with age, the error of this technique was high ( 1 14.9 years) at 65% level of confidence.
14.5 Chemical composition of teeth With age, teeth become weaker and more prone to fracture. Due to the process of aging, a noncarious transparent dentin is formed, starting at the apex of the root and sometimes extending into the coronal dentin [69 71]. The transparency of teeth is due to the mineralization of the dentin substance around the dentinal tubules (peritubular dentin) and the gradual reduction of the dentinal tubules [72]. Scanning electron microscopic studies were developed by Kosa et al. in order to analyze the finer structure of dentin associated with age. According to these studies, dentinal tubules become thinner with increasing age, although it is not significant. Also associated with aging is the widening of the intertubular area and aggregation of the peritubular matrix. All of these changes are accompanied by changes in chemical composition [73]. In 1989, Kosa et al. studied the composition of the bones using electron probe microanalysis [74], finding a significant decrease in the Ca:P ratio in older individuals. In dentin [73], they found a decrease in the Ca:P ratio with age in peritubular (hypermineralized) dentin. Also, the weight of phosphorus is more closely correlated with age, but not the amount of Ca. Raman spectroscopy is a useful tool to study the chemical composition of teeth. Tramini et al. [75] used Raman microspectrometry to analyze the dentinal part of the tooth. Four different dentinal areas were defined on each tooth, coronal dentin, dentin cementum limit, root dentin, and apical dentin. Finally, stretch bands were identified, generating a multivariate analysis model. The partial correlation coefficient between predictors and age provided mainly a high value with a mean error of 5 years. Also, the authors found slight differences between males and females. Despite these results, some predictors in the model seemed to follow a nonlinear progression with age. Raman spectroscopy coupled with ultraviolet resonance Raman spectroscopy (UVRRS) has been used by Ager et al. [76] to analyze age-induced changes in cortical bone, finding alterations in the amide I band. By applying similar techniques in human teeth [77], they also found an increase in the
204
SECTION | IV Biochemical approach of age estimation
amide I peak height in dehydrated and demineralized dentin, as a result of the increased interaction between collagen fibrils caused by stretching and intrafibrillar movement.
14.6 Advanced glycation end products The Maillard reaction is a complex series of reactions between reducing sugars and amino groups of proteins, which leads to browning, fluorescence, and cross-linking of protein [78]. AGEs, formed during the later stages of the Maillard reaction, accumulate in long-lived tissue proteins and may contribute to the development of complications in aging [79]. Human aging is associated with a stiffening of tissues that are rich in extracellular matrix and long-lived proteins, such as skeletal muscle, tendons, joints, bone, heart, arteries, lung, skin, and lens [80]. Glyoxal (GO), methylglyoxal (MGO), and deoxyglucosones belong to a series of dicarbonyl compounds, identified as intermediates in the Maillard reaction. GO and MGO react with lysine and arginine residues in protein to yield well-characterized compounds, such as the N-(carbosyalkyl)lysines, N-(carboxymethyl)lysine (CML) and N-(1-(1-carbooxy)ethyl) lysine (CEL), and imidazolones and dehydroimidazolones [81 83]. The majority of the studies related to AGEs were developed with aging purposes. They found an accumulation of AGEs in permanent and long-lived proteins during an individual’s lifetime, mainly focusing on pentosidine, CML, CEL, and/or furosine in dentin [84], crystalline lens [85], articular cartilage [86,87], and skin collagen [88]. A study conducted by Sato et al. [89] described an increase of AGEs with age in hippocampal pyramidal neurons using an antibody against AGEs to demonstrate an immunohistochemical. Although they did not find an AGE signal in young individuals, the signal increases with age, accumulating higher amounts in older individuals. The authors suggested the possibility of forensic uses of this technique in fire death cases since they found this correlation with age. Pilin et al. [90 92] demonstrated the age-related color changes in intervertebral disc excisions, Achilles tendons, and rib cartilage as a result of accumulation of AGEs. However, the color changes and correlation coefficients were different among tissues, being more conspicuous in the rib cartilage than in the intervertebral discs, and finding almost no relation with age in Achilles tendon. In addition, age estimation was only reliable up to 45 years old, beyond that the method is less accurate. The color changes caused by aging and the utilization of this effect for age estimation were studied on hard dental tissues. Some authors demonstrated that teeth become more yellow with age [93,94]. Using a spectroradiometry technique to measure these color changes, Martin-de las Heras et al. [95] tried to correlate these changes with age. Although they found a correlation, the error averaged 13.7 years. They also found differences in
Age estimation based on chemical approaches Chapter | 14
205
dental color depending on the postmortem interval. Thus, this technique is not suitable for samples with extended postmortem intervals. In a recent study, Greis et al. [96] analyzed AGEs in dentin for their applicability in forensic age estimation. They used high performance liquid chromatography (HPLC) to determine the levels of pentosidine and found a high correlation, although there was an error of 1 9.4 years. Unfortunately, this technique cannot be applied in teeth of individuals with diabetes mellitus, because AGEs are accumulated in diabetes. The same study indicated a lack of suitability for use in fire death cases, although they posited the possibility of the use of this technique in combination with aspartic acid racemization.
14.7 Discussion Among the chemical techniques presented here, aspartic acid racemization seems to be the best to apply to determine age in forensic cases. It can be used in different tissues with high accuracy, although the preferable tissue is dentin [32,39]. In addition, the technique GC is available for forensic labs, although currently only two labs apply this technique to forensic casework [13]. Despite the success of aspartic acid racemization, it is not exempt from disadvantages. It cannot be used in bodies exposed to high temperatures because the reaction is affected by temperature [31]. Also, several control teeth are needed [32,33,40], although it is possible to overcome this drawback using an artificial mixture that could be continuously available in forensic laboratories [40]. The majority of lead accumulation studies were developed for health purposes, and these studies are not in agreement with respect to the accumulation of lead with age. The only study developed in a forensic context [44] found a significant correlation between age and dentin lead levels. However, this is only applicable to Kuwaiti populations because lead levels change between geographic regions as these levels depend on industrial activity and environmental conditions. Thus, population-specific references are required to develop a formula for the estimation of age-at-death with this technique. A decrease in the reducible collagen crosslinks associated with increased age was demonstrated in different studies and tissues [67], although these studies were developed for better understanding of the aging phenomena. The only study carried out with a forensic focus [68] did find a correlation with age in dentin. However, the error on age estimation was high and the technique is time consuming. The relationship between changes in the chemical composition of teeth with age using different methodologies is less developed. The latest studies point to Raman spectroscopy as an elected technique [97]. However, it requires a wide knowledge of chemistry to correctly relate the results of dental composition with age.
206
SECTION | IV Biochemical approach of age estimation
AGEs are widely studied with aging and age-related disease purposes [78]. Forensic studies point to a correlation of AGEs with age, such as in hippocampal pyramidal neurons [89]. Other studies are based on color changes due to AGEs [90 92]; however, these studies indicate differences in the correlation between tissues. A correlation based on color was found in dentin, but the error rate was high [95]. A recent study [96] analyzed levels of pentosidine in dentin finding a high correlation with age, although the error between estimated and real age was high. In addition, this technique is not suitable for teeth from individuals with diabetes and/or in fire death cases, although this study pointed out the possibility of using this technique in conjunction with aspartic acid racemization.
References [1] Helfman PM, Bada JL. Aspartic acid racemization in tooth enamel from living humans. Proc Natl Acad Sci USA 1975;72(8):2891 4. [2] Masters PM, Bada JL, Zigler Jr. JS. Aspartic acid racemisation in the human lens during ageing and in cataract formation. Nature 1977;268(5615):71 3. [3] Helfman PM, Bada JL, Shou MY. Considerations on the role of aspartic acid racemization in the aging process. Gerontology 1977;23(6):419 25. [4] Kuhn W. Possible relation between optical activity and aging. Adv Enzymol Related Subj Biochem 1958;20:1 29. [5] Ritz S, Schutz HW. Aspartic acid racemization in intervertebral discs as an aid to postmortem estimation of age at death. J Forensic Sci 1993;38(3):633 40. [6] Helfman PM, Bada JL. Aspartic acid racemisation in dentine as a measure of ageing. Nature 1976;262(5566):279 81. [7] Ohtani S. Estimation of age from dentin by using the racemization reaction of aspartic acid. Am J Forensic Med Pathol 1995;16(2):158 61. [8] Ritz S, Schutz HW, Schwarzer B. The extent of aspartic acid racemization in dentin: a possible method for a more accurate determination of age at death? Zeitschrift fur Rechtsmedizin. J Leg Med 1990;103(6):457 62. [9] Ohtani S, Sugimoto H, Sugeno H, Yamamoto S, Yamamoto K. Racemization of aspartic acid in human cementum with age. Arch Oral Biol 1995;40(2):91 5. [10] Ohtani S. Studies on age estimation using racemization of aspartic acid in cementum. J Forensic Sci 1995;40(5):805 7. [11] Garner WH, Spector A. Racemization in human lens: evidence of rapid insolubilization of specific polypeptides in cataract formation. Proc Natl Acad Sci USA 1978;75 (8):3618 20. [12] Masters PM, Bada JL, Zigler Jr. JS. Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts. Proc Natl Acad Sci USA 1978;75(3):1204 8. [13] Klumb K, Matzenauer C, Reckert A, Lehmann K, Ritz-Timme S. Age estimation based on aspartic acid racemization in human sclera. Int J Leg Med 2016;130(1):207 11. Available from: https://doi.org/10.1007/s00414-015-1255-6. [14] Man EH, Sandhouse ME, Burg J, Fisher GH. Accumulation of D-aspartic acid with age in the human brain. Science 1983;220(4604):1407 8.
Age estimation based on chemical approaches Chapter | 14
207
[15] Dobberstein RC, Tung SM, Ritz-Timme S. Aspartic acid racemisation in purified elastin from arteries as basis for age estimation. Int J Leg Med 2010;124(4):269 75. Available from: https://doi.org/10.1007/s00414-009-0392-1. [16] Ohtani S, Matsushima Y, Kobayashi Y, Kishi K. Evaluation of aspartic acid racemization ratios in the human femur for age estimation. J Forensic Sci 1998;43(5):949 53. [17] Ohtani S, Yamamoto T, Matsushima Y, Kobayashi Y. Changes in the amount of Daspartic acid in the human femur with age. Growth Dev Aging: GDA 1998;62 (4):141 8. [18] Ohtani S. Rate of aspartic acid racemization in bone. Am J Forensic Med Pathol 1998;19 (3):284 7. [19] Ohtani S. Technical notes for age estimation using the femur: influence of various analytical conditions on D-aspartic acid contents. Int J Leg Med 2002;116(6):361 4. [20] Tiplamaz S, Goren MZ, Yurtsever NT. Estimation of chronological age from postmortem tissues based on amino acid racemization. J Forensic Sci 2018. Available from: https:// doi.org/10.1111/1556-4029.13737. [21] Matzenauer C, Reckert A, Ritz-Timme S. Estimation of age at death based on aspartic acid racemization in elastic cartilage of the epiglottis. Int J Leg Med 2014;128 (6):995 1000. Available from: https://doi.org/10.1007/s00414-013-0940-6. [22] Ohtani S, Matsushima Y, Kobayashi Y, Yamamoto T. 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. J Forensic Sci 2002;47(1):32 6. [23] Ritz S, Turzynski A, Schutz HW. Estimation of age at death based on aspartic acid racemization in noncollagenous bone proteins. Forensic Sci Int 1994;69(2):149 59. [24] Ritz S, Turzynski A, Schutz HW, Hollmann A, Rochholz G. Identification of osteocalcin as a permanent aging constituent of the bone matrix: basis for an accurate age at death determination. Forensic Sci Int 1996;77(1-2):13 26. [25] Ritz-Timme S, Cattaneo C, Collins MJ, Waite ER, Schutz HW, Kaatsch HJ, et al. Age estimation: the state of the art in relation to the specific demands of forensic practise. Int J Leg Med 2000;113(3):129 36. [26] Yekkala R, Meers C, Van Schepdael A, Hoogmartens J, Lambrichts I, Willems G. Racemization of aspartic acid from human dentin in the estimation of chronological age. Forensic Sci Int 2006;159(Suppl 1):S89 94. Available from: https://doi.org/10.1016/j. forsciint.2006.02.022. [27] Ohtani S, Yamamoto T. 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. J Forensic Sci 2005;50(5):1020 7. [28] Yekkala R, Meers C, Hoogmartens J, Lambrichts I, Willems G, Van Schepdael A. An improved sample preparation for an LC method used in the age estimation based on aspartic acid racemization from human dentin. J Sep Sci 2007;30(1):118 21. [29] Ohtani S, Yamamoto K. Estimation of ages from racemization of an amino acid in teeth—assessment of errors under various experimental conditions. Nihon hoigaku zasshi 1991;45(2):124 7. [30] Ohtani S, Yamamoto K. Age estimation using the racemization of amino acid in human dentin. J Forensic Sci 1991;36(3):792 800. [31] Ritz S, Schutz HW, Peper C. Postmortem estimation of age at death based on aspartic acid racemization in dentin: its applicability for root dentin. Int J Leg Med 1993;105 (5):289 93.
208
SECTION | IV Biochemical approach of age estimation
[32] Sakuma A, Ohtani S, Saitoh H, Iwase H. Comparative analysis of aspartic acid racemization methods using whole-tooth and dentin samples. Forensic Sci Int 2012;223(13):198 201. Available from: https://doi.org/10.1016/j.forsciint.2012.08.043. [33] Ohtani S, Yamamoto K. Estimation of age from a tooth by means of racemization of an amino acid, especially aspartic acid—comparison of enamel and dentin. J Forensic Sci 1992;37(4):1061 7. [34] Griffin RC, Moody H, Penkman KE, Collins MJ. The application of amino acid racemization in the acid soluble fraction of enamel to the estimation of the age of human teeth. Forensic Sci Int 2008;175(1):11 16. Available from: https://doi.org/10.1016/j. forsciint.2007.04.226. [35] Sirin N, Matzenauer C, Reckert A, Ritz-Timme S. Age estimation based on aspartic acid racemization in dentine: what about caries-affected teeth? Int J Leg Med 2018;132 (2):623 8. Available from: https://doi.org/10.1007/s00414-017-1667-6. [36] Ohtani S. Different racemization ratios in dentin from different locations within a tooth. Growth Dev Aging: GDA 1997;61(2):93 9. [37] Ohtani S, Yamada Y, Yamamoto I. Improvement of age estimation using amino acid racemization in a case of pink teeth. Am J Forensic Med Pathol 1998;19(1):77 9. [38] Sakuma A, Saitoh H, Ishii N, Iwase H. The effects of racemization rate for age estimation of pink teeth. J Forensic Sci 2015;60(2):450 2. Available from: https://doi.org/10.1111/ 1556-4029.12653. [39] Ohtani S, Yamamoto T. Age estimation by amino acid racemization in human teeth. J Forensic Sci 2010;55(6):1630 3. Available from: https://doi.org/10.1111/j.15564029.2010.01472.x. [40] Ohtani S, Abe I, Yamamoto T. An application of D- and L-aspartic acid mixtures as standard specimens for the chronological age estimation. J Forensic Sci 2005;50 (6):1298 302. [41] Ritz-Timme S, Rochholz G, Schutz HW, Collins MJ, Waite ER, Cattaneo C, et al. Quality assurance in age estimation based on aspartic acid racemisation. International J Leg Med 2000;114(1-2):83 6. [42] Ohtani S, Yamagishi M, Ogasawara M, Yamamoto K. Age estimation of two unidentified bodies by amino acid racemization in their teeth. Bull Kanagawa Dent Coll: BKDC/KDS 1990;18(1):23 7. [43] Ohtani S, Utsunomiya J, Minoshima T, Yamamoto K. Tooth-based age estimation of an adipocerated cadaver using the amino acid racemization method. Nihon hoigaku zasshi 1994;48(4):279 81. [44] Al-Qattan SI, Elfawal MA. Significance of teeth lead accumulation in age estimation. J Forensic Legal Med 2010;17(6):325 8. Available from: https://doi.org/10.1016/j. jflm.2010.05.001. [45] Steenhout A, Pourtois M. Lead accumulation in teeth as a function of age with different exposures. Br J Ind Med 1981;38(3):297 303. [46] Fosse G, Justesen NP. Lead in deciduous teeth of Norwegian children. Arch Environ Health 1978;33(4):166 75. [47] Haavikko K, Anttila A, Helle A, Vuori E. Lead concentrations of enamel and dentine of deciduous teeth of children from two Finnish towns. Arch Environ Health 1984;39 (2):78 84. [48] Bercovitz K, Laufer D. Lead accumulation in teeth of patients suffering from gastrointestinal ulcers. Sci Total Environ 1991;101(3):229 34.
Age estimation based on chemical approaches Chapter | 14
209
[49] Bercovitz K, Laufer D. Age and gender influence on lead accumulation in root dentine of human permanent teeth. Arch Oral Biol 1991;36(9):671 3. [50] Drasch GA, Ott J. Lead in human bones. Investigations on an occupationally non-exposed population in southern Bavaria (F.R.G.). II. Children. Sci Total Environ 1988;68:61 9. [51] Somervaille LJ, Chettle DR, Scott MC. In vivo measurement of lead in bone using x-ray fluorescence. Phys Med Biol 1985;30(9):929 43. [52] Nusbaum RE, Butt EM, Gilmour TC, Didio SL. Relation of air pollutants to trace metals in bone. Arch Environ Health 1965;10:227 32. [53] Altshuller LF, Halak DB, Landing BH, Kehoe RA. Deciduous teeth as an index of body burden of lead. J Pediatr 1962;60:224 9. [54] Mackie AC, Stephens R, Townshend A. Tooth lead levels in Birmingham children. Arch Environ Health 1977;32(4):178 85. [55] Pinchin MJ, Newham J, Thompson RP. Lead, copper, and cadmium in teeth of normal and mentally retarded children. Clin Chim Acta 1978;85(1):89 94. [56] Habercam JW, Keil JE, Reigart JR, Croft HW. Lead content of human blood, hair, and deciduous teeth: correlation with environmental factors and growth. J Dent Res 1974;53 (5):1160 3. [57] Holtzman RB, Lucas Jr. HF, Ilcewicz FH. The concentration of lead in human bone. ANL-7615. ANL Rep 1968;43 9. [58] Strehlow CD, Kneip TJ. The distribution of lead and zinc in the human skeleton. Am Ind Hyg Assoc J 1969;30(4):372 8. Available from: https://doi.org/10.1080/00028896909343140. [59] Bercovitz K, Laufer D. Lead levels in teeth of long date and new immigrants in several cities in Israel—preliminary results. Public Health Rev 1991;19(1-4):141 6. [60] Eyre D. Collagen cross-linking amino acids. Methods Enzymol 1987;144:115 39. [61] Mechanic G, Gallop PM, Tanzer ML. The nature of crosslinking in collagens from mineralized tissues. Biochem Biophys Res Commun 1971;45(3):644 53. [62] Piez KA. Cross-linking of collagen and elastin. Annu Rev Biochem 1968;37:547 70. Available from: https://doi.org/10.1146/annurev.bi.37.070168.002555. [63] Eyre DR, Paz MA, Gallop PM. Cross-linking in collagen and elastin. Annu Rev Biochem 1984;53:717 48. Available from: https://doi.org/10.1146/annurev.bi.53.070184.003441. [64] Bailey AJ, Shimokomaki MS. Age related changes in the reducible cross-links of collagen. FEBS Lett 1971;16(2):86 8. [65] Moriguchi T, Fujimoto D. Age-related changes in the content of the collagen crosslink, pyridinoline. J Biochem 1978;84(4):933 5. [66] Eyre DR, Dickson IR, Van Ness K. Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues. Biochem J 1988;252(2):495 500. [67] Walters C, Eyre DR. Collagen crosslinks in human dentin: increasing content of hydroxypyridinium residues with age. Calcif Tissue Int 1983;35(4-5):401 5. [68] Martin-de las Heras S, Valenzuela A, Villanueva E. Deoxypyridinoline crosslinks in human dentin and estimation of age. Int J Leg Med 1999;112(4):222 6. [69] Micheletti Cremasco M. Dental histology: study of aging processes in root dentine. Bollettino della Societa italiana di biologia sperimentale 1998;74(3-4):19 28. [70] Vasiliadis L, Darling AI, Levers BG. The histology of sclerotic human root dentine. Arch Oral Biol 1983;28(8):693 700. [71] Vasiliadis L, Darling AI, Levers BG. The amount and distribution of sclerotic human root dentine. Arch Oral Biol 1983;28(7):645 9.
210
SECTION | IV Biochemical approach of age estimation
[72] Amprino R, Engstrom A. Studies on x ray absorption and diffraction of bone tissue. Acta Anat 1952;15(1-2):1 22. [73] Kosa F, Antal A, Farkas I. Electron probe microanalysis of human teeth for the determination of individual age. Med Sci Law 1990;30(2):109 14. [74] Kosa F, Farkas I, Wittman G. [Microprobe study of bones in the determination of individual age]. Morphologiai es igazsagugyi orvosi szemle 1989;29(3):227 32. [75] Tramini P, Bonnet B, Sabatier R, Maury L. A method of age estimation using Raman microspectrometry imaging of the human dentin. Forensic Sci Int 2001;118(1):1 9. [76] Ager JW, Nalla RK, Breeden KL, Ritchie RO. Deep-ultraviolet Raman spectroscopy study of the effect of aging on human cortical bone. J Biomed Opt 2005;10(3):034012. Available from: https://doi.org/10.1117/1.1924668. [77] Ager 3rd JW, Nalla RK, Balooch G, Kim G, Pugach M, Habelitz S, et al. On the increasing fragility of human teeth with age: a deep-UV resonance Raman study. J Bone Miner Res 2006;21(12):1879 87. Available from: https://doi.org/10.1359/jbmr.060816. [78] Baynes JW. The role of AGEs in aging: causation or correlation. Exp Gerontol 2001;36 (9):1527 37. [79] Thorpe SR, Baynes JW. Role of the Maillard reaction in diabetes mellitus and diseases of aging. Drugs Aging 1996;9(2):69 77. [80] Monnier VM, Mustata GT, Biemel KL, Reihl O, Lederer MO, Zhenyu D, et al. Crosslinking of the extracellular matrix by the maillard reaction in aging and diabetes: an update on “a puzzle nearing resolution”. Ann N Y Acad Sci 2005;1043:533 44. Available from: https://doi.org/10.1196/annals.1333.061. [81] Ahmed MU, Brinkmann Frye E, Degenhardt TP, Thorpe SR, Baynes JW. N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem J 1997;324(Pt 2):565 70. [82] Ahmed MU, Thorpe SR, Baynes JW. Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein. J Biol Chem 1986;261 (11):4889 94. [83] Henle T, Walter AW, Klostermeyer H. A simple method for the preparation of furosine and pyridosine reference material. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung 1994;198(1):66 7. [84] Miura J, Nishikawa K, Kubo M, Fukushima S, Hashimoto M, Takeshige F, et al. Accumulation of advanced glycation end-products in human dentine. Arch Oral Biol 2014;59(2):119 24. Available from: https://doi.org/10.1016/j.archoralbio.2013.10.012. [85] Marques C, Ramalho JS, Pereira P, Mota MC. Bendazac decreases in vitro glycation of human lens crystallins. Decrease of in vitro protein glycation by bendazac. Doc Ophthalmol 1995;90(4):395 404. [86] Verzijl N, DeGroot J, Ben ZC, Brau-Benjamin O, Maroudas A, Bank RA, et al. Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: a possible mechanism through which age is a risk factor for osteoarthritis. Arthritis Rheum 2002;46(1):114 23 doi:10.1002/1529-0131 (200201)46:1 , 114::AID-ART10025 . 3.0.CO;2-P. [87] Verzijl N, DeGroot J, Oldehinkel E, Bank RA, Thorpe SR, Baynes JW, et al. Age-related accumulation of Maillard reaction products in human articular cartilage collagen. Biochem J 2000;350(Pt 2):381 7. [88] Dyer DG, Dunn JA, Thorpe SR, Bailie KE, Lyons TJ, McCance DR, et al. Accumulation of Maillard reaction products in skin collagen in diabetes and aging. J Clin Invest 1993;91 (6):2463 9. Available from: https://doi.org/10.1172/JCI116481.
Age estimation based on chemical approaches Chapter | 14
211
[89] Sato Y, Kondo T, Ohshima T. Estimation of age of human cadavers by immunohistochemical assessment of advanced glycation end products in the hippocampus. Histopathology 2001;38(3):217 20. [90] Pilin A, Pudil F, Bencko V. The image analysis of colour changes of different human tissues in the relation to the age. Part 2: practical applicability. Soudni lekarstvi 2007;52 (3):36 42. [91] Pilin A, Pudil F, Bencko V. The image analysis of colour changes of different human tissues in the relation to the age. Part 1. Methodological approach. Soudni lekarstvi / casopis Sekce soudniho lekarstvi Cs lekarske spolecnosti J Ev Purkyne 2007;52(2):26 30. [92] Pilin A, Pudil F, Bencko V. Changes in colour of different human tissues as a marker of age. Int J Leg Med 2007;121(2):158 62. Available from: https://doi.org/10.1007/s00414006-0136-4. [93] Ten Cate AR, Thompson GW, Dickinson JB, Hunter HA. The estimation of age of skeletal remains from the colour of roots of teeth. Dent J 1977;43(2):83 6. [94] Lackovic KP, Wood RE. Tooth root colour as a measure of chronological age. J Forensic Odonto-Stomatol 2000;18(2):37 45. [95] Martin-de las Heras S, Valenzuela A, Bellini R, Salas C, Rubino M, Garcia JA. Objective measurement of dental color for age estimation by spectroradiometry. Forensic Sci Int 2003;132(1):57 62. [96] Greis F, Reckert A, Fischer K, Ritz-Timme S. Analysis of advanced glycation end products (AGEs) in dentine: useful for age estimation? Int J Leg Med 2017. Available from: https://doi.org/10.1007/s00414-017-1671-x. [97] Heudt L, Debois D, Zimmerman TA, Kohler L, Bano F, Partouche F, et al. Raman spectroscopy and laser desorption mass spectrometry for minimal destructive forensic analysis of black and color inkjet printed documents. Forensic Sci Int 2012;219(1-3):64 75. Available from: https://doi.org/10.1016/j.forsciint.2011.12.001.