Estimation of age at death based on aspartic acid racemization in noncollagenous bone proteins

Forensic SCbllC8

Forensic Science International 69 (1994) 149-159

ELSEVIER

Estimation of age at death based on aspartic acid racemization in noncollagenous bone proteins S. Ritz*“,

A. Turzynskib,

H.W.

Schiitza

alnstitul ftir Rechtstnedizin der Christian-Albrechts- Universitiit zu Eel. Arnold-Heller-Sir. 12, D-24105 Kiel, Germany blnstitut fir Pathologic der Humboldt-Universitii zu Berlin, Schumannsir. 20/21. D-10117 Berlin, Germany

Received 15 August 1993; accepted 23 September 1994

Abstract

Age at death determination based on the extent of aspartic acid racemization in dentin has been reported to be highly accurate and reproducible. To test the applicability of this method to human bone, aspartic acid racemization in noncollagenous proteins of bone was investigated. A close relationship was found between age at death and the extent of aspartic acid racemization in osteocalcin, the most abundant noncollagenous protein of the organic bone matrix. Our findings indicate that osteocalcin is a permanent, ‘aging’ constituent of the organic bone matrix whose D-aspartic acid content increases with age because of in vivo racemization. Thus, the extent of aspartic acid racemization in bone osteocalcin is a measure of the age of the peptide and hence of the entire organism. The relationship between age at death and the extent of aspartic acid racemization in purified bone osteocalcin appears to be close enough to serve as a basis for determination of age at death in forensic medicine. Keywords: Age at death determination;

Aspartic acid racemization; Non-collagenous

proteins

of bone; Osteocalcin

1. Introduction During aging, a gradual transformation of L-aspartic acid into its D-form (racemization) occurs in many tissues of the human organism, including dentin, tooth enamel, lens, white matter of the brain, lung and intervertebral discs [l-21]. * Corresponding author. 0379-0738/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0379-0738(94)01659-S

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In vivo racemization may produce an age-dependent increase in the D-aspartic acid concentration in tissues containing metabolically stable, permanent proteins that were synthesized early in life and not subsequently exchanged. For several years now, estimation of age at death based on in vivo racemization of aspartic acid in dentin has been applied successfully in forensic odontology [ 13- 17,19,20]. The organic matrix of dentin consists of approximately 9 1% collagen and 9% noncollagenous material [22]. Following acid extraction, the acid insoluble dentinal protein fraction is composed chiefly of collagen, while noncollagenous proteins and peptides predominate in the acid soluble fraction [9,23]. Aspartic acid racemization is rapid in (acid soluble) noncollagenous proteins or peptides of dentin but proceeds only slowly or not at all in the (acid insoluble) collagenous protein fraction [8,9,15,16,19]. With advancing age, the D-aspartic acid content of dentin increases, predominantly in the noncollagenous protein fraction [8,9,15, 16,191. A number of studies have shown that age at death determination based on aspartic acid racemization in dentinal protein is more exact and of superior reproducibility than most other techniques for postmortem estimation of age [ 13- 17,19,20]. Teeth, however, are not always available in forensic practice. In many instances, age at death has to be determined by the analysis of bone. The organic matrix of bone consists of 90% insoluble collagen and 10% noncollagenous protein [24,25]; it is very similar to that of dentin [22]. Some noncollagenous proteins, osteocalcin for example, occur in both of these calcified tissues [26-311. Consequently, bone - like dentin - should contain metabolically stable, noncollagenous proteins with an age-dependent accumulation of D-aSpartiC acid. If this is true, age at death determination based on aspartic acid racemization should be applicable to bone. To test this supposition, the extent of aspartic acid racemization in the total noncollagenous (acid soluble) bone protein fraction, and in different noncollagenous proteins of bone was determined.

2. Materials and methods 2.1. Determination

of the extent of aspartic acid racemization collagenous (acid soluble) bone protein fraction

in the total non-

2.1.1. Preparation of specimens Bone specimens were taken from 35 human skulls (postmortem intervals up to 8 days). After removal of the soft tissue, the specimens were freeze dried and pulverized. Contaminants were removed by stirring the pulverized bone overnight in a 15% NaCl solution, for 15 min in ethanol/ether (3: 1) and for 1 h in 2% sodium dodecyl sulfate. To avoid in vitro proteolysis, all washing steps were carried out at 4°C; moreover, wash solutions (except ethanol/ether) contained protease inhibitors (2.5 mM benzamidine HCl, 50 mM e-amino-n-caproic acid, 0.5 mM N-ethylmaleimide, and 0.3 mM phenylmethylsulfonylfluoride). After washing, the specimens were rinsed in water and freeze dried.

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2.1.2. Acid extraction

Each 1 g of washed and freeze dried bone was extracted with 5 ml 20% formic acid at 4°C for 3 h. The supematant containing the acid soluble bone proteins was dried in a vacuum. 2.1.3. Determination of the extent of aspartic acid racemization The extent of aspartic acid racemization was determined as described elsewhere [19]. Briefly, the dried acidic extracts were hydrolysed for 6 h in 6 N HCl at 100°C. The hydrolysate was esteritied with isopropanol/sulfuric acid (10: 1) for 1 h at 110°C. After alkaline extraction with dichloromethane, acetylation was performed with trifluoracetic anhydride (TFA) at 60°C for 15 min. The amino acids were now present as TFA-isopropylesters. D- and L-aspartic acid were separated and quantified by gas chromatography on a chiral capillary column (Chirasil-Val) using a flame ionization detector, with hydrogen as carrier gas. 2.1.4. Evaluation

The racemization of amino acids can be described as [32-341: ln [(l + D/L)/(l- D/L)]= 2 k(Asp.) t + constant where D/L represents the proportion of D-aspartic acid to L-aspartic acid, k(Asp.) is the first-order rate constant of the interconversion of enantiomers, and t equals time. The value In [( 1 + D/L)/(1 - D/L)]was calculated for each sample. The relationship between age at death and the extent of aspartic acid racemization (In [(l + D/ ~)/(l- D/L)])was evaluated by linear regression analysis. 2.2. Determination

of the extent of aspartic acid racemization collagenous bone proteins separated by gel filtration

in different

non-

2.2. I. Extraction of noncollagenous bone proteins Skull bone was prepared as described above (2.1 .l.). Each 10 g of washed and freeze dried bone was extracted with 50 ml 20% formic acid at 4°C for 3 h. The extracts were desalted on columns of Sephadex G-25 M (PD-10 columns, Pharmacia), equilibrated in 20% formic acid, and freeze dried. 2.2.2. Gel filtration

The freeze dried acidic extracts were dissolved in 4 M guanidine HCl, 0.05 M Tris-HCl, pH 8.0 and applied to a column Sephacryl S-200 HR (65 x 2.6 cm; Pharmacia) equilibrated with the same buffer at 20°C. Elution occurred at a flow rate of 30 ml/h. Fractions of 5 ml were collected. 2.2.3. Determination fractions

of the extent of aspartic acid racemization

in all gel filtration

The gel filtration fractions were desalted (on Sephadex G-25 M, PD-10 columns

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Pharmacia, equilibrated in 20% formic acid) and freeze dried. The extent of aspartic acid racemization was determined in all gel filtration fractions of 10 individuals with different ages at death (16,21,28, 36, 39,45, 52,65, 71 and 82 years). Measurements and evaluation were done as described above (Sections 2.1.3. and 2.1.4.). 2.2.4. Determination of osteocalcin immunoreactivity in gel filtration fractions by an enzyme-linked immunosorbant assay (ELISA) Relative osteocalcin concentrations in the gel filtration fractions were estimated using a noncompetitive solid-phase enzyme immunoassay procedure and a bridged detection method. Assays were performed in triplicate. Gel filtration fractions were desalted on columns of Sephadex G-25 M (PD-10 columns, Pharmacia) equilibrated in 0.1 M Tris-HCl, pH 8.0. The sample antigen was immobilized on a polystyrene microtiter plate (Maxisorb, Nunc, Germany) by adding 5 ~1 of each desalted fraction to 95 ~120 mM potassium carbonate buffer (pH 9.5) and was allowed to bind to the plate overnight at 4°C. All subsequent incubation steps were performed at 37°C. Each incubation was terminated by washing three times with 0.05% Tween-20 in phosphate-buffered saline (PBS-Dulbecco; 137 mM NaCl, 2.7 mM KCl, 1.44 mM KH2P04, 5.18 mM Na2HP04, pH 7.4). After binding of the antigen, the remaining nonspecific binding sites on the plate were blocked by coating with 2% bovine serum albumin (BSA) in PBS. The monoclonal primary antibody directed against the 21-31 residue of osteocalcin (Pierce, USA) was diluted 1:1000 in a solution of 1% BSA, 0.05% Tween in PBS and was allowed to react for 2 h. An affinity purified biotinylated rabbit-anti-mouse antibody (Dako, Germany) diluted 1:1000 in the same buffer served as secondary antibody. The sensivitity was increased by introducing a biotinylated goat-anti-rabbit antibody (Dako) as tertiary antibody. Both layers

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0

10

20

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60

70

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90

Age (years) Fig. 1.Extent of aspartic acid racemization (In [( 1+D/L)/( 1 - D/L)]) in the total noncollagenous (acid soluble) bone protein fraction in relation to age at death (n = 35). The regression line is shown for ages at death below 60 years (n = 25; r = 0.96).

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were incubated for 1 h. Streptavidine horseradish peroxidase complexes (Dako) prepared according to the supplier’s instruction (Dako) were applied for 45 min in a dilution of 1:100. Peroxidase activity was determined calorimetrically using 3.3’,5.5’-tetramethylbenzidine as substrate. Absorbances were read at 450 nm on a microplate reader. 3. Results 3.1. Extent of aspartic acid racemization in the total noncollagenous (acid soluble) bone protein fraction The extent of aspartic acid racemization in the acid extracted, noncollagenous bone protein fraction increased with age (Fig. 1). A relatively close relationship between age at death and the extent of aspartic acid racemization was found only below an age at death of about 60 years (r = 0.96; Fig. 1). In cases with high ages at death, this relationship became poor and the extent of aspartic acid racemization tended to be low (Fig. 1). 3.2. Extent of aspartic acid racemization in different noncollagenous bone proteins separated by gel filtration Fig. 2 presents a typical gel filtration chromatogram of the acidic extract of one bone and the extent of aspartic acid racemization in each gel filtration fraction. The different proteins contained in the acidic extract of a single bone exhibited different

r

10

20

30

40 Fraction

50

60

70

Number

Fig. 2. Typical Sephacryl S-200 HR gel filtration chromatogram (solid line) and extent of aspartic acid racemization (In [(I + D/L)/ (I - D/L)]; columns) in all gel filtration fractions of a single bone (each column represents the mean value of two adjacent fractions; fractions indicated by asterisks did not contain substantial amounts of aspartic acid).

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J -0

10

20

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40

50

60

70

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90

Age (years)

Fig. 3. Extent of aspartic acid racemization (In [(I + D/L)/(l - D/L)]) in the gel filtration fractions 38-42 (see bar in Fig. 4) of 10 different bone specimens in relation to age at death (r = 0.99).

D-aspartic acid contents. With decreasing molecular weight, the extent of aspartic acid racemization increased, reaching a maximum in the peptides with lowest molecular weights. Analysis of all gel filtration fractions of 10 different bone specimens revealed a close relationship between age at death and the extent of aspartic acid racemization

10

20

40

30 Fraction

50

60

70

Number

Fig. 4. Elution position of osteocalcin in a typical Sephacryl S-200 HR gel filtration chromatogram (solid line), as determined by an ELISA (broken line). Osteocalcin eluted as a sharp peak predominantly in the gel filtration fractions 38-42 (bar); these fractions exhibited a close relationship between age at death and the extent of aspartic acid racemization (see Fig. 3).

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only in the major peak eluting around fraction 40 (Fig. 3). In this peak, the bulk of osteocalcin eluted, as demonstrated by an ELISA for this peptide (Fig. 4). 4. Discussion Demineralization of dentin or bone by acid extraction releases noncollagenous proteins. In the noncollagenous, acid soluble protein of dentin, a close relationship exists between dentin age and the extent of aspartic acid racemization; this can serve as a basis for a highly accurate estimation of age at death [ l&16,19]. The results presented here demonstrate that racemization of aspartic acid occurs also in acid soluble, noncollagenous proteins of bone; as in dentin, the extent of aspartic acid racemization in acid extracted, noncollagenous bone proteins increased with age (Fig. 1). However, the relationship between age and the extent of aspartic acid racemization in total acidic extracts of bone became poorer with increasing age (Fig. 1) and was generally poorer than that reported for dentinal extracts [8,9,15,16,19]. Total acidic extracts of bone contain several noncollagenous proteins. The major constituents of the noncollagenous bone protein fraction are osteocalcin, osteonectin and sialoproteins; other components are proteoglycans, matrix gla protein (MGP), bone morphogenic protein (BMP) and non-bone-specific serum proteins, such as q-HS glycoprotein, albumin, transferrin and IgG [24,27,28,35-371. Most of these proteins can be easily extracted by acid extraction. Gel filtration of acidic extracts of bone separates the extracted proteins according to their molecular weight. In the gel filtration fractions of a single bone, the extent of aspartic acid racemization tended to increase with decreasing molecular weight of the noncollagenous bone proteins and peptides (Fig. 2). Very similar results have been reported for fossil bones and marine sediments; the analyzed amino acids racemized slowly in proteins, faster in peptides, and still faster in free amino acids [32,33,38,39]. Thus, the rate of amino acid racemization depends upon the physicochemical environment in which the amino acids are contained [6,32-34,38,39]. Every protein or peptide seems to have its own kinetics of aspartic acid racemization. Therefore, the extent of aspartic acid racemization in the total acidic extracts of bone is determined not only by the age of the analyzed bone, but also by the composition of the extracts. Since several noncollagenous proteins of bone degrade with increasing age [24], the composition of acidic extracts of bone changes with age. The inconstant, agedependent composition of total acidic bone extracts appears to be the reason for the poor relationship between age at death and the extent of aspartic acid racemization in the extracts from individuals of advanced age (Fig. 1). Thus, total acidic extracts of bone are not suitable for an exact determination of age at death based on aspartic acid racemization because of their variable composition. In contrast to the results for total acidic bone extracts, a very close relationship between age at death and the extent of aspartic acid racemization was evident in certain gel filtration fractions (fractions 38-42, Figs. 3 and 4), when all gel filtration fractions of the acidic extracts of 10 bones of different ages were analyzed. In these gel filtration fractions the noncollagenous bone peptide osteoculcin eluted, as demonstrated by an ELISA (Fig. 4). Despite the small number of specimens analyz-

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ed (n = lo), our data strongly indicate an age-related accumulation of Daspartic acid in osteocalcin due to in vivo racemization of aspartic acid in this noncollagenous bone peptide. Osteocalcin is apparently the most abundant of the noncollagenous matrix proteins of bone, accounting for about lo-20% of the total [40-451. It appears,to be a universal constituent of the skeleton and tooth dentin of all vertebrates and is one of the 10 most abundant proteins in the entire organism [28,42,43,45-471. Human osteocalcin consists of 49 amino acids, is synthesized by osteoblasts and odontoblasts and binds strongly to hydroxyapatite [28,29,40,41,44,47-491. The biological function of osteocalcin is still unknown; possible roles in matrix formation or as an ‘informational molecule’ in Ca2+ homeostasis have been discussed [28,41,44,47,49]. Osteocalcin first appears in developing embryonic bone, coincident with the onset of bone mineralization [28,41,47,49]. Adult levels are reached in human bone long before birth [47,49]. The half-life of osteocalcin is not yet known, but osteocalcin has been thought to be a permanent element of the extracellular bone matrix with a half-life of several years [48,50]. Determination of aspartic acid racemization in a definite, purified protein enables a tissue-specific evaluation of its turnover [21]; a close relationship between the extent of aspartic acid racemization and age at death is only possible if the analyzed protein was synthesized early in life and not subsequently exchanged [21]. The data reported here, therefore, indicate that osteocalcin is a permanent constituent of the organic bone matrix that ‘ages’with the organism. Accordingly, the extent of aspartic acid racemization in purified bone osteocalcin may be used as a measure of the age of the peptide and hence of the entire organism. The relationship between age at death and the extent of aspartic acid racemization in @rifled bone osteocalcin appears to be close enough to serve as a basis for determination of age at death (Fig. 3). Even in cases with long postmortem intervals, purification and analysis of bone osteocalcin should be possible since osteocalcin has even been detected in fossil bones [28]. Of course, estimating age at death by determining the extent of aspartic acid racemization in purified bone osteocalcin is methodically demanding and requires the appropriate laboratory equipment. However, in light of the accuracy obtained in estimating age based on aspartic acid racemization in dentin [ 13,14,16,19,20], our findings on aspartic acid racemization in bone osteocalcin promise a new biochemical method for determining age at death that may be superior in precision and reproducibility to most other methods applicable to bone. Thus, future efforts will be devoted to the optimization of the method by developing additional purification steps to ensure the purity of the analyzed peptide while simplifying the laboratory technique. Moreover, the accuracy and reproducibility of the method must be further evaluated by analysis of a sufficiently large number of bone specimens from different sites. The data presented here, together with the findings of other authors on aspartic acid racemization in the human organism [l-21], permit the following conclusions: (1)

Aging of the human organism involves not only cells, but also permanent, extracellular proteins, which are synthesized early in life and not exchanged

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(2)

(3)

(4)

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during life. Osteocalcin, one of the most abundant noncollagenous proteins of the organic bone matrix, appears to be such a permanent and ‘aging’ protein. In several permanent proteins, such as noncollagenous dentinal proteins [8,9,15,16,19], lung elastin f21] and bone osteocalcin, an age-dependent accumulation of D-aspartic acid occurs because of in vivo racemization of aspartic acid. The extent of aspartic acid racemization in purified bone osteocalcin and in other permanent proteins is a measure of the aging of these proteins and hence of the entire organism. In vivo racemization of aspartic acid is a universal phenomenon which occurs not only in dentin and bone, but also in other tissues [l-4,6,7,10-12,18,21]. The identification of additional permanent and ‘aging’ proteins of the human organism may provide further insights into biological functions and open up new possibilities for the determination of age at death in forensic medicine.

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