INTRODUCTION
Human skeletal remains are commonly used in forensic anthropology to identify of unknown individuals (Konigsberg et al. 2008) particularly in cases where the remains are presented in the form of fragmentation, burning or severe damage from other causes such as mass disasters, fires or crimes (Owsley et al. 1985). The data of skeletal remains have established a biological profile that can be compared to unidentified individuals. The biological profile consists of sex, age, ancestry and stature. These data are very useful variables for narrowing the range of identification (Blau & Briggs 2011; Cattaneo 2007). Moreover, the data from finger prints, dental data and DNA analysis provide the most accuracy for specific individuals. But these methods take a long time to analyze data and are expensive (Carolan et al. 1997) or few countries lack DNA database. Thus, the 169information from biological profile is first usually applied in forensic context for primary identification (Qudsia et al. 2014).
The various methods for age estimation are based on the assessment of developmental and degenerative changes in the skeletons such as macroscopic morphological studies (Albert 1998; Brooks 1955), histomorphometry method (Inthasan & Mahakkanukrauh 2017; Kerley 1965), radiological assessment (Martin et al. 1981), and biochemical method (Helfman & Bada 1975; Helfman & Bada 1976). There are several biochemical processes in protein which is related to determining age such as oxidation, isomerization and racemization process (Balin & Allen 1989; Stadtman 1988). Racemization of amino acid method has been generally used in forensic sciences because this analysis process is more objective and accuracy (Ogino et al. 1985; Ohtani 1995b; Ohtani & Yamamoto 2010). AMINO ACID
Amino acid is the building block of proteins in which one amino acid connects to another one to become chains of polypeptide. Many polypeptides are connected together to protein. Generally, chemical form of amino acid is composed of an amino group (NH3), a carboxyl group (COOH), a hydrogen atom (H), and R group or side chain which relies on the type of amino acids. The four different groups or atom bond to an alpha carbon atom in the centre (Johnson & Miller 1997; McCudden & Kraus 2006). Amino acids combine with other molecules by peptide bond which forms between amino group of one amino acid and the carboxyl end. Amino acids within organism proteins have important role in many systems in the body such as structure of cells, transportation and storage, formation of enzymes or antibodies (Robins et al. 2001). Amino acids commonly present in two forms which are L-(levorotary) and D-(dextrorotary) forms. These forms are enantiomers that mean both L- and D-forms are mirror image of each other and is not superimposed like the right and left hands. L-amino acids use in protein synthesis (Poinar et al. 1996). The chemical property of two forms is not different (Bada 1982). Amino acids within the proteins initially form in the L-form and during aging the L- form of amino acids will be gradually converted to D-form (Iida et al. 2001). There is accumulation of D-form increased with age. Therefore, interconversion of both forms amino acids involves organism age (Holtkötter 2011; Johnson & Miller 1997). This process is called amino acid racemization.
AMINO ACID RACEMIZATION
Racemization is the reversible first-order reaction of amino acids that is the transformation of L- and D-form amino acids. This reaction takes place in the tissues with low metabolic turnover rate at body temperature in mammalian (Masters et al. 1978; Ohtani et al. 2002). This process is depended on temperature, time, pH (Fisher et al. 1986). Age estimation from low metabolism tissues provide more precise than high metabolism tissues because in tissues with high metabolic rate have continually form L-amino acids. So, there is not formation of D-amino acids. The change D/L ratio of amino acid during aging can be used to estimate the death time of the organism. The low protein turnover tissue which is usually used for amino acid racemization is teeth and racemization rate is approximately 0.1% per year in the low metabolism tissue like dentine (Csapo et al. 1994). Moreover, racemization can be occurred in various structures such as the bones, white matter of brain, artery and eye-lens (Dobberstein et al. 2010; Griffin et al. 2008a; Johnson & Miller 1997). The previous study (Hare & Abelson 1968) examined amino acids in fossils shell. They reported that D-amino acids were obtained from transformation of L-amino acid and the amount of D-amino acids was continually increased with age. Furthermore, there was higher the D/L ratio of amino acids in older fossil shells. Then, there were application of amino acid racemization in fossils to estimated age by measurement the D/L ratio of amino acids (Hare & Mitterer 1969). Consequently, amino acid racemization is widely applied for age estimation in many tissues which comprise proteins. Furthermore, the relationship between amino acids racemization and age can be used for investigation of lifetime of protein ageing (Helfman et al. 1977) and pathological diseases in older individuals (Powell et al. 1992; Shapiro et al. 1991).
ASPARTIC ACID RACEMIZATION
There are about 20 general amino acids in human body that divided into essential and non-essential amino acids (Ouellette & Rawn 2014). Aspartic acid (Asp) is non-essential amino acids in the body which commonly used for racemization in age estimation. Racemization of most amino acids is slow process but the accumulation of D-aspartic acids is most rapidly of all stable amino acids in the bones and other tissues (Robins et al. 2001; Yekkala et al. 2006). Aspartic acid racemization has rate constants at neutral pH (Poinar et al. 2006) and can occur throughout lifetime and after death, as well (Yekkala et al. 2006). There was a report about racemization rate of many amino acids in fossil and it was found that racemization rate of aspartic acid was highest when compared to other amino acids (Ohtani et al. 2002). Aspartic acid has weak peptide bond in protein chain so, it has rapid racemization rate (Goodfriend et al. 1992). Rate of racemization is one of the important factors for accuracy of age estimation. Since aspartic acid has the highest rate of racemization, there is adequate amount of D-aspartic acids for amino acid analysis. Thus, aspartic acid is the most suitable indicator of age determination (Ohtani et al. 2002).
Racemization of aspartic acid in polypeptide chain relates a cyclic succinimide intermediate or succinyl residue (Asu) (Van Duin & Collins 1998). Hydrolysis of aspartic residue will be produced a succinmide intermediate (Robins et al. 2001) via the process is called nucleophilic attack. Several of the sites of suucinimide intermediate formation usually occur in the terminal regions of proteins. The process initiate from the nitrogen of the carbon terminal peptide bond attract to the carbon molecule of carboxylic acid group creating the conformation of unstable sucinimide ring or called cyclization. This succinimide ring is short lived structure so, it has rapid hydrolysis. Because of this metastable property of succinimide ring, the ring is susceptibility. Thus, the ring structure can be transformed from L-aspartic acid into D-aspartic acid rapidly. In addition, formation of D-aspartic acids also involves asparagines (Asn) by deamidation process (McCudden & Kraus 2006; Ritz-Timme & Collins 2002). Additionally, the racemization rate of aspartic acid in peptide bond is due to reaction of succinimide intermediate formation (Geiger & Clarke 1987; Radkiewicz et al. 1996). FACTORS TO RACEMIZATION
Amino acid racemization is the chemical reaction which depends on many factors mentioned below.
TEMPERATURE AND PH
Temperature has numerous effects to the ratio of D/L amino acids because racemization is chemical reaction. According to the previous study, they investigated racemization of amino acid in dental enamel. The result found that molars were slightly higher temperature than other teeth because these teeth were located on the back regions in the mouth. Racemization of molars teeth was more rapid than incisors which cooler temperature (Ohtani et al. 2005). This shows that the higher the temperature causes fast racemization rate. Therefore, the specimens should be preserved at low temperatures (below atmospheric temperature) like cadavers preserve at lower temperatures before using for analysis (McCudden & Kraus 2006; Ohtani & Yamamoto 2005).
For pH factor is supported by the previous report; the authors studied aspartic acid racemization in human dentin. They stored the teeth in different solution include acidic, alkaline, distilled water, and in dry conditions. The results showed that the rate of racemization was highest in pH 9 solutions and followed by in distilled water, in pH 4 solution, and in dry condition, respectively. Furthermore, the estimated age from teeth in these environment for 1 year demonstrated that there were slightly increased by about 0.007 years in a dry condition. On the other hand, the teeth left in the pH 9 conditions showed increase of 0.6 years. It is indicated that in alkaline condition is more effective compare to other conditions (Ohtani 1995a).
HUMIDITY
In wet condition, rate of racemization is higher than dry condition. So, the cadavers left in high humidity condition like a tropical rain forest may be not suitable for racemization analysis (Ohtani et al. 2005; Ohtani & Yamamoto 2005).
FIXATION
In another study applied heating experiment was done to determine racemization in teeth of human cadavers. They stored the teeth in different fixatives which were 95% ethanol, 10% formalin solution and 10% neutral formalin fixative at several temperatures. The rate of aspartic acid racemization was highest in 10% neutral formalin solution, followed by in 10% formalin solution, and in 95% ethanol, respectively. Nevertheless, the teeth stored at 15