Int J Legal Med DOI 10.1007/s00414-013-0940-6
ORIGINAL ARTICLE
Estimation of age at death based on aspartic acid racemization in elastic cartilage of the epiglottis Christian Matzenauer & Alexandra Reckert & Stefanie Ritz-Timme
Received: 2 August 2013 / Accepted: 30 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Age estimation based on aspartic acid racemization (AAR) has been applied successfully to various tissues. For routine uses, AAR is analyzed in dentine. For cases in which teeth are unavailable, analyzing AAR in purified elastin has been shown to be an alternative method. The suitability of elastic cartilage from the epiglottis as an elastin source for age estimation based on AAR was tested. A total of 65 tissue samples (cartilage) of epiglottis and 45 samples of elastin purified from the elastic cartilage of epiglottis samples were analyzed. While the D -aspartic acid content of total tissue samples increased with age only slowly, its increase with age in purified elastin samples was similar to that in purified elastin from other tissues. The relationship between the D aspartic acid content and age was shown to be close enough for age estimation based on AAR in purified elastin from the elastic cartilage of the epiglottis, provided a sufficient quality of elastin purification. Age estimation based on AAR in purified elastin from the epiglottis might serve as a valuable alternative in cases in which other tissues (e.g., teeth) are unavailable. Keywords Age estimation . Aspartic acid racemization . Epiglottis . Elastic cartilage . Elastin
Introduction Accurate estimation of age at death is crucial for the identification of unknown deceased persons. One of the most accurate methods for age estimation in adults is based on the racemization of aspartic acid (AAR) [1]. C. Matzenauer (*) : A. Reckert : S. Ritz-Timme Institute of Forensic Medicine, University Clinic, Heinrich Heine University, Moorenstr. 5, 40225 Duesseldorf, Germany e-mail: [email protected]
The method and its molecular basis have already been described in detail [2–4]. Permanent proteins with low or no turnover may exhibit an accumulation of D -amino acids with increasing age. The resulting age-dependent increase of the D aspartic acid content with a close relationship between age and the extent of aspartic acid racemization is the basis of age estimation based on AAR [5]. Age estimation based on AAR can be applied to purified permanent proteins or to tissues containing permanent proteins in constant and relevant concentrations. The method has been successfully applied to total tissue samples without protein purification like intervertebral discs [6], to crude protein extracts of dentine [7–12], enamel [13], and cementum [14] from teeth, to various fractions of cortical bone [15, 16] and of alveolar bone [17], to various protein fractions of rib cartilage [18], as well as to purified proteins like osteocalcin from bone [19] and elastin from skin [20], from arteries [21], and from ligamenta flava [22]. The most accurate results were achieved by determining the ratio of D -aspartic acid to L -aspartic acid in tissues with a constant protein composition like dentine [7, 8, 10–12] and in purified long-living proteins like osteocalcin [19] and elastin [20]. Methods for age estimation based on AAR in the permanent “aging” elastin have been described for ligamenta flava [22], arteries [21], and skin [20]. Basically, all these tissues can be analyzed successfully. However, the purification of elastin from the different tissues is of different complexity; the processing of the samples is much more sophisticated in the case of skin and artery tissue than in the analysis of ligamenta flava which consist mainly of elastic fibers, with elastin being their major component [23]. Elastic cartilage is another tissue rich in elastic fibers [24]. Elastic cartilage samples can easily be obtained by preparation of the epiglottis. We tested the suitability of total tissue
Int J Legal Med
samples as well as of purified elastin samples from elastic cartilage of the epiglottis for age estimation based on AAR.
Methods Preparation of samples The tissue samples of epiglottis were collected during autopsy. Sixty-five samples of cartilage from individuals aged between 16 and 92 years were taken for analysis as total tissue samples, and 45 samples from individuals aged between 24 and 96 years were prepared for analysis of purified elastin. Samples were taken from bodies without signs of putrefaction or signs of exposure to heat only. The whole epiglottis except for one piece kept for histological examination (measuring around 0.5×0.2×0.1 cm) was used for analysis. After the mucosa had been pulled off, avoiding contamination with blood at all times, the total tissue samples were then cut in small pieces and washed in 15 % sodium chloride solution containing protease inhibitors (2.5 mM benzamidine HCl, 50 mM ε-amino-η-caproic acid, 0.5 mM N ethylmaleimide, and 0.3 mM phenylmethylsulfonyl fluoride) at 4 °C for 24 h. After rinsing in bidistilled water at 4 °C, the samples were defatted for 15 min using a solution of three parts ethanol and one part diethyl ether. Then they were rinsed in bidistilled water at 4 °C again. After that, the samples were freeze-dried and stored at −20 °C until further procedures.
24 h in 6 N HCl at 110 °C, amino acid analyses by HPLC were performed on an Agilent 1100 Series (Agilent Technologies, Böblingen, Germany). Primary and secondary amino acids were detected in a single run due to a combined pre-column derivatization with OPA/MPA (o -phthaldialdehyde/3-mercaptopropionic acid) and FMOC (9-fluorenylmethylchloroformate). Amino acids were separated on a Hypersil BDS C18 250×3 mm, 5 μm column (Thermo Electron GmbH, Dreieich, Germany) using a flow of 1 ml min−1 at 40 °C and a gradient from 90 % mobile phase A (40 mM sodium dihydrogen phosphate monohydrate (NaH2PO4), 1.5 mM sodium azide (NaN3), adjusted to pH 7.8), and 10 % mobile phase B (450 ml methanol, 450 ml acetonitrile, 100 ml water) from 100 to 43 % within 40 min. After the run, the column was flushed with 100 % B for 7 min, followed by a 6-min equilibration at a flow rate of 1.2 ml min−1. Primary amino acids were detected at an excitation wavelength of 340 nm and a detection wavelength of 450 nm. Secondary amino acids (proline, hydroxyproline) were detected at an excitation wavelength of 340 nm and a detection wavelength of 266 and 305 nm. For signal identification and quantification, calibrated external standards were used. As the content of hydroxyproline in the purified elastin samples indicates the amount of contaminating collagen, the hydroxyproline content of the samples was taken as a parameter for the quality of elastin purification. As glycine is the amino acid most abundant in elastin, it was used as a denominator for the hydroxyproline content.
Histology Of each sample, one piece was histologically examined in regard to fibrous proliferation and degradation of elastic fibers (hematoxylin–eosin and Elastica van Gieson stains). Purification of elastin The samples taken for elastin purification were then digested twice with bacterial collagenase (Sigma type 7, 250 U 100 mg−1 tissue in 1.25 ml 50 mmol L−1 of tricine buffer with 10 mmol L−1 calcium chloride and 400 mmol L−1 sodium chloride at pH 7.5 at 25 °C) for 24 h each at 37 °C. The residues were then washed with bidistilled water and freeze-dried. Assessing the quality of elastin purification After elastin purification, one piece of each sample was stained with Elastica van Gieson for histological examination. Furthermore, the quality of the elastin purification was biochemically assessed by amino acid analysis of the purified elastin samples and an elastin standard (elastin from bovine neck ligament, Sigma). After hydrolysis of the samples for
Determination of the extent of AAR The freeze-dried samples of total tissue and of purified elastin, respectively, were hydrolyzed for 6 h in 6 N HCl at 100 °C. Hydrochloric acid and water were then removed in a vacuum. The hydrolysate was esterified with isopropanol/sulfuric acid (10:1) for 1 h at 110 °C. After neutralization with ammonia, dichloromethane was added, and the samples were centrifugalized. After extracting the phase containing dichloromethane, acetylation using trifluoroacetic anhydride (TFAA) was done for 15 min at 60 °C. The amino acids were now present as TFA-isopropyl esters. The ratio of D -aspartic acid to L -aspartic acid was measured after separation of the amino acids by gas chromatography on a Shimadzu GC-2014, using a chiral capillary column and a flame ionization detector. Hydrogen was used as carrier gas. Statistics The relationship between AAR and age was evaluated by inverse prediction, using a simple linear regression analysis [25].
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the epiglottis. The relation between AAR (ln ((1+D /L )/ (1−D /L ))) and age (t ) in years can be described by the linear regression equation: lnðð1 þ D=LÞ=ð1−D=LÞÞ ¼ 0:0011t þ 0:0577;
Results
with the correlation coefficient r =0.76 and the standard error of regression s y.x =0.02057. In higher ages, relationship between AAR in total tissue samples and age was relatively weak. Figure 3 excludes individuals aged over 60 years and shows the extent of AAR in relation to age at death in 35 total tissue samples only of individuals aged 60 and below. The relationship between AAR (ln ((1+D /L )/(1−D /L ))) and age (t ) in years in this age group can be described by the linear regression equation:
Quality of tissue samples and elastin purification
lnðð1 þ D=LÞ=ð1−D=LÞÞ ¼ 0:0017t þ 0:0349;
After histological examination of 65 total tissue samples, nine of them were excluded due to the histological detection of a fibrous proliferation and/or of a marked degradation of elastic fibers (see Fig. 1 for an example of marked fibrous and fatty proliferation). At least a mild degradation of elastic cartilage could be seen histologically in virtually all of the older individuals. The purified elastin samples appeared to be free from contaminating collagen fibers histologically. The amino acid composition of the purified elastin samples exhibited the typical amino acid profile of elastin. However, the hydroxyproline content of the samples indicated relevant collagen contaminations in at least some samples. The hydroxproline/glycine quotient was above 3 % in 13 samples and even above 5 % in two samples.
with the correlation coefficient r =0.87 and the standard error of regression s y.x =0.01231.
Fig. 1 Histological section of an epiglottis sample showing marked fibrous and fatty proliferation (Elastica van Gieson stain)
AAR in total tissue samples Figure 2 depicts the extent of AAR in relation to age at death in 56 total tissue samples of elastic cartilage of Fig. 2 Extent of AAR in relation to age at death in 56 total tissue samples of elastic cartilage of the epiglottis
AAR in purified elastin samples Figure 4 depicts the extent of AAR in relation to age at death in 45 samples of purified elastin from elastic cartilage. The relationship between AAR (ln ((1+D /L )/(1−D/L ))) and age (t) in years can be described by the linear regression equation: lnðð1 þ D=LÞ=ð1−D=LÞÞ ¼ 0:0037t þ 0:0353; with the correlation coefficient r =0.84 and the standard error of regression s y.x =0.04361. The relationship between AAR in the purified elastin and age is evenly close in younger and older ages. However, samples with a high hydroxyproline/glycine quotient (as indication of a contamination by collagen) caused a higher scattering of data.
0.45 0.4 0.35 0.3 total tissue samples
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Int J Legal Med Fig. 3 Extent of AAR in relation to age at death in 35 total tissue samples of elastic cartilage of the epiglottis: only individuals aged 60 and below
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After exclusion of two samples exhibiting a hydroxyproline/ glycine quotient higher than 5 %, the relationship between AAR and age was significantly closer. The relationship between AAR (ln ((1+D/L)/(1−D/L))) and age (t) in years in this group can be described by the linear regression equation: lnðð1 þ D=LÞ=ð1−D=LÞÞ ¼ 0:0038t þ 0:0311; with the correlation coefficient r =0.89 and the standard error of regression s y.x =0.03751. After excluding 13 more samples showing a hydroxyproline/glycine quotient higher than 3 %, the relationship between AAR and age was even closer. The relationship between AAR (ln ((1+D /L )/(1−D/L ))) and age (t) in years can be described by the linear regression equation: lnðð1 þ D=LÞ=ð1−D=LÞÞ ¼ 0:0040t þ 0:0213; with the correlation coefficient r =0.92 and the standard error of regression s y.x =0.03538. Fig. 4 Extent of AAR in relation to age at death in 45 samples of purified elastin from elastic cartilage of the epiglottis
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Discussion AAR in total tissue samples of elastic cartilage of the epiglottis Total tissue samples of elastic cartilage of the epiglottis exhibited an increase of aspartic acid racemization with age (Fig. 2), thereby confirming that elastic cartilage from the epiglottis contains permanent or at least long-living proteins that age with the organism. Although it could be assumed that the main permanent protein contributing to the accumulation of D aspartic acid with age in the analyzed samples is elastin, the increase of the D -aspartic acid content with age in the total tissue samples of elastic cartilage of the epiglottis was relatively low compared to the increase of the D -aspartic acid content found in purified elastin from various other tissues [20–22]. This points to a considerable content of “contaminating” proteins without intravital racemization of amino acids, first of all to contaminating collagen that does not exhibit a relevant racemization of its aspartic acid residues [26, 27]. The relationship between AAR and age in the elastic cartilage samples is quite close in younger ages; with increasing
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age, the relationship is poorer (Fig. 2). This is a typical finding for total tissue samples or crude protein extracts in case of protein degradation and tissue remodeling increasing with age, resulting in very different protein compositions in higher ages (see data for bone [2, 16, 19]). Quality of elastin purification Although we used a simplified (and thus very practical) method for elastin purification, the purified samples exhibited the typical amino acid composition of elastin. However, the hydroxyproline/glycine quotients of some samples indicated at least slight contaminations with collagen. Even slight contaminations are crucial for age estimation based on AAR, since collagen contains large amounts of non-racemized aspartic acid residues [26, 27]. AAR in purified elastin from elastic cartilage of the epiglottis Purified elastin samples exhibited a steeper increase of the aspartic acid content with age and a closer relationship between AAR and age at death (Fig. 4) than total tissue samples. The kinetics of aspartic acid racemization found in purified elastin from the elastic cartilage from epiglottis was similar to that described for purified elastin from other tissues [20–22]. Although the relationship between AAR and age in purified elastin samples lacking major collagen contamination was close (r =0.92), it was worse than in purified elastin from other tissues (e.g., skin, r =0.99 [20]; ligamenta flava, r = 0.96–0.99 [22]). This could be in part due to a sometimes incomplete removal of collagen by the simplified purification process employed, indicated by higher hydroxyproline/ glycine quotients. Exclusion of samples showing a high amount of hydroxyproline markedly improved the correlation of the extent of AAR and of the age at death. Samples showing a Hyp/Gly quotient of more than 5 % are not optimally suitable for age estimation in forensic practice; results of the analysis of such samples should be interpreted very carefully. Age estimation based on AAR in elastic cartilage of the epiglottis The applicability of a method for age estimation in forensic practice depends on the achievable accuracy of age diagnosis and on the practicability of the analytical steps needed. The accuracy of age estimation based on AAR in elastic cartilage of the epiglottis remains to be analyzed by the investigation of an independent sample collection. Furthermore, the accuracy of the method in cases with signs of putrefaction or exposure to heat has to be tested. The achievable accuracy in cases without signs of putrefaction or heat exposure is roughly indicated by the calculated correlation coefficients. It is
obviously lower than the achievable accuracy of age estimation based on AAR in dentine [7, 8, 10–12], but should be higher than that of most other methods for age estimation in adults [1, 28]. The practicability of the method is comparable to that of age estimation based on AAR in dentine. It is an interesting alternative method for cases in which teeth are not available for age estimation based on AAR.
References 1. Ritz-Timme S, Cattaneo C, Collins MJ, Waite ER, Schütz HW, Kaatsch H-J, Borrman HIM (2000) Age estimation: the state of the art in relation to the specific demands of forensic practise. Int J Legal Med 113:129–136 2. Ritz-Timme S (1999) Lebensaltersbestimmung aufgrund des Razemisierungsgrades von Asparaginsäure: Grundlagen, Methodik, Möglichkeiten, Grenzen, Anwendungsbereiche. In: Berg S, Brinkmann B (eds) Arbeitsmethoden der medizinischen und naturwissenschaftlichen Kriminalistik, Bd. 23. Verlag Schmidt Römhild, Lübeck 3. Ritz-Timme S, Rochholz G, Schütz HW, Collins MJ, Waite ER, Cattaneo C, Kaatsch H-J (2000) Quality assurance in age estimation based on aspartic acid racemisation. Int J Legal Med 114:83–86 4. Stephenson RC, Clarke S (1989) Succinimide formation from aspartyl and asparaginyl peptides as a model for the spontaneous degradation of proteins. J Biol Chem 264:6164–6170 5. Ritz-Timme S, Collins MJ (2002) Racemization of aspartic acid in human proteins. Ageing Res Rev 1:43–59 6. Ritz S, Schütz HW (1993) Aspartic acid racemization in intervertebral discs as an aid to postmortem estimation of age at death. J Forensic Sci 38:633–640 7. Ohtani S, Yamamoto K (1991) Age estimation using the racemization of amino acid in human dentin. J Forensic Sci 36:792–800 8. Ritz S, Schütz HW, Peper C (1993) Postmortem estimation of age at death based on aspartic acid racemization in dentin: its applicability for root dentin. Int J Legal Med 105:289–293 9. Ohtani S (1994) Age estimation by aspartic acid racemization in dentin of deciduous teeth. Forensic Sci Int 68:77–82 10. Ohtani S (1995) Estimation of age from dentin by using the racemization reaction of aspartic acid. Am J Forensic Med Pathol 16:158–161 11. Ritz S, Stock R, Schütz HW, Kaatsch HJ (1995) Age estimation in biopsy specimens of dentin. Int J Legal Med 108:135–139 12. Fu S-J, Fan C-C, Song H-W, Wie F-Q (1995) Age estimation using a modified HPLC determination of ratio of aspartic acid in dentin. Forensic Sci Int 73:35–40 13. Ohtani S, Yamamoto K (1992) 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 37:1061–1067 14. Ohtani S, Sugimoto H, Sugeno H, Yamamoto S, Yamamoto K (1995) Racemization of aspartic acid in human cementum with age. Arch Oral Biol 40:91–95 15. Pfeiffer H, Mörnstad H, Teivens A (1995) Estimation of chronologic age using the aspartic acid racemization method. II. On human cortical bone. Int J Legal Med 108:24–26 16. Ritz S, Turzynski A, Schütz HW (1994) Estimation of age at death based on aspartic acid racemization in noncollagenous bone proteins. Forensic Sci Int 69:149–159 17. Ohtani S, Yamamoto T, Abe I, Kinoshita Y (2007) Age-dependent changes in the racemisation ratio of aspartic acid in human alveolar bone. Arch Oral Biol 52:233–236
Int J Legal Med 18. Pfeiffer H, Mörnstad H, Teivens A (1995) Estimation of chronologic age using the aspartic acid racemization method. I. On human rib cartilage. Int J Legal Med 108:19–23 19. Ritz S, Turzynski A, Schütz HW, Hollmann A, Rochholz G (1996) Identification of osteocalcin as a permanent aging constituent of the bone matrix: basis for an accurate age at death determination. Forensic Sci Int 77:13–26 20. Ritz-Timme S, Laumeier I, Collins MJ (2003) Aspartic acid racemization: evidence for marked longevity of elastin in human skin. Br J Dermatol 149:951–959 21. Dobberstein RC, Tung S-M, Ritz-Timme S (2010) Aspartic acid racemisation in purified elastin from arteries as basis for age estimation. Int J Legal Med 124:269–275 22. Ritz-Timme S, Laumeier I, Collins M (2003) Age estimation based on aspartic acid racemization in elastin from the yellow ligaments. Int J Legal Med 117:96–101
23. Viejo-Fuertes D, Liguoro D, Rivel J, Midy D, Guerin J (1998) Morphologic and histologic study of the ligamentum flavum in the thoraco-lumbar region. Surg Radiol Anat 20: 171–176 24. Mecham RP, Heuser JE (1991) The elastic fiber. In: Hay ED (ed) Cell biology of extracellular matrix, 2nd edn. Plenum, New York 25. Zar JH (1984) Biostatistical analysis, 2nd edn. Prentice Hall, Eaglewood Cliffs 26. van Duin ACT, Collins MJ (1998) The effects of conformational constraints on aspartic acid racemization. Org Geochem 29:1227– 1232 27. Collins MJ, Waite ER, van Duin AC (1999) Predicting protein decomposition: the case of aspartic-acid racemization kinetics. Philos Trans R Soc Lond B Biol Sci 354:51–64 28. Meissner C, Ritz-Timme S (2010) Molecular pathology and age estimation. Forensic Sci Int 203:34–43
ORIGINAL ARTICLE
Estimation of age at death based on aspartic acid racemization in elastic cartilage of the epiglottis Christian Matzenauer & Alexandra Reckert & Stefanie Ritz-Timme
Received: 2 August 2013 / Accepted: 30 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Age estimation based on aspartic acid racemization (AAR) has been applied successfully to various tissues. For routine uses, AAR is analyzed in dentine. For cases in which teeth are unavailable, analyzing AAR in purified elastin has been shown to be an alternative method. The suitability of elastic cartilage from the epiglottis as an elastin source for age estimation based on AAR was tested. A total of 65 tissue samples (cartilage) of epiglottis and 45 samples of elastin purified from the elastic cartilage of epiglottis samples were analyzed. While the D -aspartic acid content of total tissue samples increased with age only slowly, its increase with age in purified elastin samples was similar to that in purified elastin from other tissues. The relationship between the D aspartic acid content and age was shown to be close enough for age estimation based on AAR in purified elastin from the elastic cartilage of the epiglottis, provided a sufficient quality of elastin purification. Age estimation based on AAR in purified elastin from the epiglottis might serve as a valuable alternative in cases in which other tissues (e.g., teeth) are unavailable. Keywords Age estimation . Aspartic acid racemization . Epiglottis . Elastic cartilage . Elastin
Introduction Accurate estimation of age at death is crucial for the identification of unknown deceased persons. One of the most accurate methods for age estimation in adults is based on the racemization of aspartic acid (AAR) [1]. C. Matzenauer (*) : A. Reckert : S. Ritz-Timme Institute of Forensic Medicine, University Clinic, Heinrich Heine University, Moorenstr. 5, 40225 Duesseldorf, Germany e-mail: [email protected]
The method and its molecular basis have already been described in detail [2–4]. Permanent proteins with low or no turnover may exhibit an accumulation of D -amino acids with increasing age. The resulting age-dependent increase of the D aspartic acid content with a close relationship between age and the extent of aspartic acid racemization is the basis of age estimation based on AAR [5]. Age estimation based on AAR can be applied to purified permanent proteins or to tissues containing permanent proteins in constant and relevant concentrations. The method has been successfully applied to total tissue samples without protein purification like intervertebral discs [6], to crude protein extracts of dentine [7–12], enamel [13], and cementum [14] from teeth, to various fractions of cortical bone [15, 16] and of alveolar bone [17], to various protein fractions of rib cartilage [18], as well as to purified proteins like osteocalcin from bone [19] and elastin from skin [20], from arteries [21], and from ligamenta flava [22]. The most accurate results were achieved by determining the ratio of D -aspartic acid to L -aspartic acid in tissues with a constant protein composition like dentine [7, 8, 10–12] and in purified long-living proteins like osteocalcin [19] and elastin [20]. Methods for age estimation based on AAR in the permanent “aging” elastin have been described for ligamenta flava [22], arteries [21], and skin [20]. Basically, all these tissues can be analyzed successfully. However, the purification of elastin from the different tissues is of different complexity; the processing of the samples is much more sophisticated in the case of skin and artery tissue than in the analysis of ligamenta flava which consist mainly of elastic fibers, with elastin being their major component [23]. Elastic cartilage is another tissue rich in elastic fibers [24]. Elastic cartilage samples can easily be obtained by preparation of the epiglottis. We tested the suitability of total tissue
Int J Legal Med
samples as well as of purified elastin samples from elastic cartilage of the epiglottis for age estimation based on AAR.
Methods Preparation of samples The tissue samples of epiglottis were collected during autopsy. Sixty-five samples of cartilage from individuals aged between 16 and 92 years were taken for analysis as total tissue samples, and 45 samples from individuals aged between 24 and 96 years were prepared for analysis of purified elastin. Samples were taken from bodies without signs of putrefaction or signs of exposure to heat only. The whole epiglottis except for one piece kept for histological examination (measuring around 0.5×0.2×0.1 cm) was used for analysis. After the mucosa had been pulled off, avoiding contamination with blood at all times, the total tissue samples were then cut in small pieces and washed in 15 % sodium chloride solution containing protease inhibitors (2.5 mM benzamidine HCl, 50 mM ε-amino-η-caproic acid, 0.5 mM N ethylmaleimide, and 0.3 mM phenylmethylsulfonyl fluoride) at 4 °C for 24 h. After rinsing in bidistilled water at 4 °C, the samples were defatted for 15 min using a solution of three parts ethanol and one part diethyl ether. Then they were rinsed in bidistilled water at 4 °C again. After that, the samples were freeze-dried and stored at −20 °C until further procedures.
24 h in 6 N HCl at 110 °C, amino acid analyses by HPLC were performed on an Agilent 1100 Series (Agilent Technologies, Böblingen, Germany). Primary and secondary amino acids were detected in a single run due to a combined pre-column derivatization with OPA/MPA (o -phthaldialdehyde/3-mercaptopropionic acid) and FMOC (9-fluorenylmethylchloroformate). Amino acids were separated on a Hypersil BDS C18 250×3 mm, 5 μm column (Thermo Electron GmbH, Dreieich, Germany) using a flow of 1 ml min−1 at 40 °C and a gradient from 90 % mobile phase A (40 mM sodium dihydrogen phosphate monohydrate (NaH2PO4), 1.5 mM sodium azide (NaN3), adjusted to pH 7.8), and 10 % mobile phase B (450 ml methanol, 450 ml acetonitrile, 100 ml water) from 100 to 43 % within 40 min. After the run, the column was flushed with 100 % B for 7 min, followed by a 6-min equilibration at a flow rate of 1.2 ml min−1. Primary amino acids were detected at an excitation wavelength of 340 nm and a detection wavelength of 450 nm. Secondary amino acids (proline, hydroxyproline) were detected at an excitation wavelength of 340 nm and a detection wavelength of 266 and 305 nm. For signal identification and quantification, calibrated external standards were used. As the content of hydroxyproline in the purified elastin samples indicates the amount of contaminating collagen, the hydroxyproline content of the samples was taken as a parameter for the quality of elastin purification. As glycine is the amino acid most abundant in elastin, it was used as a denominator for the hydroxyproline content.
Histology Of each sample, one piece was histologically examined in regard to fibrous proliferation and degradation of elastic fibers (hematoxylin–eosin and Elastica van Gieson stains). Purification of elastin The samples taken for elastin purification were then digested twice with bacterial collagenase (Sigma type 7, 250 U 100 mg−1 tissue in 1.25 ml 50 mmol L−1 of tricine buffer with 10 mmol L−1 calcium chloride and 400 mmol L−1 sodium chloride at pH 7.5 at 25 °C) for 24 h each at 37 °C. The residues were then washed with bidistilled water and freeze-dried. Assessing the quality of elastin purification After elastin purification, one piece of each sample was stained with Elastica van Gieson for histological examination. Furthermore, the quality of the elastin purification was biochemically assessed by amino acid analysis of the purified elastin samples and an elastin standard (elastin from bovine neck ligament, Sigma). After hydrolysis of the samples for
Determination of the extent of AAR The freeze-dried samples of total tissue and of purified elastin, respectively, were hydrolyzed for 6 h in 6 N HCl at 100 °C. Hydrochloric acid and water were then removed in a vacuum. The hydrolysate was esterified with isopropanol/sulfuric acid (10:1) for 1 h at 110 °C. After neutralization with ammonia, dichloromethane was added, and the samples were centrifugalized. After extracting the phase containing dichloromethane, acetylation using trifluoroacetic anhydride (TFAA) was done for 15 min at 60 °C. The amino acids were now present as TFA-isopropyl esters. The ratio of D -aspartic acid to L -aspartic acid was measured after separation of the amino acids by gas chromatography on a Shimadzu GC-2014, using a chiral capillary column and a flame ionization detector. Hydrogen was used as carrier gas. Statistics The relationship between AAR and age was evaluated by inverse prediction, using a simple linear regression analysis [25].
Int J Legal Med
the epiglottis. The relation between AAR (ln ((1+D /L )/ (1−D /L ))) and age (t ) in years can be described by the linear regression equation: lnðð1 þ D=LÞ=ð1−D=LÞÞ ¼ 0:0011t þ 0:0577;
Results
with the correlation coefficient r =0.76 and the standard error of regression s y.x =0.02057. In higher ages, relationship between AAR in total tissue samples and age was relatively weak. Figure 3 excludes individuals aged over 60 years and shows the extent of AAR in relation to age at death in 35 total tissue samples only of individuals aged 60 and below. The relationship between AAR (ln ((1+D /L )/(1−D /L ))) and age (t ) in years in this age group can be described by the linear regression equation:
Quality of tissue samples and elastin purification
lnðð1 þ D=LÞ=ð1−D=LÞÞ ¼ 0:0017t þ 0:0349;
After histological examination of 65 total tissue samples, nine of them were excluded due to the histological detection of a fibrous proliferation and/or of a marked degradation of elastic fibers (see Fig. 1 for an example of marked fibrous and fatty proliferation). At least a mild degradation of elastic cartilage could be seen histologically in virtually all of the older individuals. The purified elastin samples appeared to be free from contaminating collagen fibers histologically. The amino acid composition of the purified elastin samples exhibited the typical amino acid profile of elastin. However, the hydroxyproline content of the samples indicated relevant collagen contaminations in at least some samples. The hydroxproline/glycine quotient was above 3 % in 13 samples and even above 5 % in two samples.
with the correlation coefficient r =0.87 and the standard error of regression s y.x =0.01231.
Fig. 1 Histological section of an epiglottis sample showing marked fibrous and fatty proliferation (Elastica van Gieson stain)
AAR in total tissue samples Figure 2 depicts the extent of AAR in relation to age at death in 56 total tissue samples of elastic cartilage of Fig. 2 Extent of AAR in relation to age at death in 56 total tissue samples of elastic cartilage of the epiglottis
AAR in purified elastin samples Figure 4 depicts the extent of AAR in relation to age at death in 45 samples of purified elastin from elastic cartilage. The relationship between AAR (ln ((1+D /L )/(1−D/L ))) and age (t) in years can be described by the linear regression equation: lnðð1 þ D=LÞ=ð1−D=LÞÞ ¼ 0:0037t þ 0:0353; with the correlation coefficient r =0.84 and the standard error of regression s y.x =0.04361. The relationship between AAR in the purified elastin and age is evenly close in younger and older ages. However, samples with a high hydroxyproline/glycine quotient (as indication of a contamination by collagen) caused a higher scattering of data.
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After exclusion of two samples exhibiting a hydroxyproline/ glycine quotient higher than 5 %, the relationship between AAR and age was significantly closer. The relationship between AAR (ln ((1+D/L)/(1−D/L))) and age (t) in years in this group can be described by the linear regression equation: lnðð1 þ D=LÞ=ð1−D=LÞÞ ¼ 0:0038t þ 0:0311; with the correlation coefficient r =0.89 and the standard error of regression s y.x =0.03751. After excluding 13 more samples showing a hydroxyproline/glycine quotient higher than 3 %, the relationship between AAR and age was even closer. The relationship between AAR (ln ((1+D /L )/(1−D/L ))) and age (t) in years can be described by the linear regression equation: lnðð1 þ D=LÞ=ð1−D=LÞÞ ¼ 0:0040t þ 0:0213; with the correlation coefficient r =0.92 and the standard error of regression s y.x =0.03538. Fig. 4 Extent of AAR in relation to age at death in 45 samples of purified elastin from elastic cartilage of the epiglottis
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Discussion AAR in total tissue samples of elastic cartilage of the epiglottis Total tissue samples of elastic cartilage of the epiglottis exhibited an increase of aspartic acid racemization with age (Fig. 2), thereby confirming that elastic cartilage from the epiglottis contains permanent or at least long-living proteins that age with the organism. Although it could be assumed that the main permanent protein contributing to the accumulation of D aspartic acid with age in the analyzed samples is elastin, the increase of the D -aspartic acid content with age in the total tissue samples of elastic cartilage of the epiglottis was relatively low compared to the increase of the D -aspartic acid content found in purified elastin from various other tissues [20–22]. This points to a considerable content of “contaminating” proteins without intravital racemization of amino acids, first of all to contaminating collagen that does not exhibit a relevant racemization of its aspartic acid residues [26, 27]. The relationship between AAR and age in the elastic cartilage samples is quite close in younger ages; with increasing
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age, the relationship is poorer (Fig. 2). This is a typical finding for total tissue samples or crude protein extracts in case of protein degradation and tissue remodeling increasing with age, resulting in very different protein compositions in higher ages (see data for bone [2, 16, 19]). Quality of elastin purification Although we used a simplified (and thus very practical) method for elastin purification, the purified samples exhibited the typical amino acid composition of elastin. However, the hydroxyproline/glycine quotients of some samples indicated at least slight contaminations with collagen. Even slight contaminations are crucial for age estimation based on AAR, since collagen contains large amounts of non-racemized aspartic acid residues [26, 27]. AAR in purified elastin from elastic cartilage of the epiglottis Purified elastin samples exhibited a steeper increase of the aspartic acid content with age and a closer relationship between AAR and age at death (Fig. 4) than total tissue samples. The kinetics of aspartic acid racemization found in purified elastin from the elastic cartilage from epiglottis was similar to that described for purified elastin from other tissues [20–22]. Although the relationship between AAR and age in purified elastin samples lacking major collagen contamination was close (r =0.92), it was worse than in purified elastin from other tissues (e.g., skin, r =0.99 [20]; ligamenta flava, r = 0.96–0.99 [22]). This could be in part due to a sometimes incomplete removal of collagen by the simplified purification process employed, indicated by higher hydroxyproline/ glycine quotients. Exclusion of samples showing a high amount of hydroxyproline markedly improved the correlation of the extent of AAR and of the age at death. Samples showing a Hyp/Gly quotient of more than 5 % are not optimally suitable for age estimation in forensic practice; results of the analysis of such samples should be interpreted very carefully. Age estimation based on AAR in elastic cartilage of the epiglottis The applicability of a method for age estimation in forensic practice depends on the achievable accuracy of age diagnosis and on the practicability of the analytical steps needed. The accuracy of age estimation based on AAR in elastic cartilage of the epiglottis remains to be analyzed by the investigation of an independent sample collection. Furthermore, the accuracy of the method in cases with signs of putrefaction or exposure to heat has to be tested. The achievable accuracy in cases without signs of putrefaction or heat exposure is roughly indicated by the calculated correlation coefficients. It is
obviously lower than the achievable accuracy of age estimation based on AAR in dentine [7, 8, 10–12], but should be higher than that of most other methods for age estimation in adults [1, 28]. The practicability of the method is comparable to that of age estimation based on AAR in dentine. It is an interesting alternative method for cases in which teeth are not available for age estimation based on AAR.
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