INTRODUCTION
Identification of human remains has been a long practice in forensic practice (Franklin 2010). When an unknown person cannot be identified, the first step for general identification process concerns the biological profile which is assessed from sex, ethnicity, stature and age (Lynnerup 2013). The estimation of age at death is one of important work of any forensic investigator. Age estimation conduce to establish a biological profile to compare with any missing person in forensic circumstances (Cunha et al. 2009). Each individual of the similar chronological age may be has difference of morphological appearance of age related skeleton (Zioupos et al. 2014). Therefore, age estimation techniques 178depend on the variation methods and skill of the specialist (Dobberstein et al. 2010). However, there are still high accuracy methods in age estimation which complicate the procedure more. One of these methods which has been accomplished in age estimation is amino acid racemization.
AMINO ACID RACEMIZATION
Amino acids are vital components in living organisms and are subunit structure of all proteins. The amino acid structure is composed of a carbon atom (C) which has four different groups include an amino group (-NH2), a carboxyl group (-COOH), a hydrogen atom (-H), andside chain or Rgroup (-R). Side chain is different part of amino acids that depends upon the kind of amino acids. There are twenty types of amino acids which are present in various tissues and bones. Amino acid is linked to one another with peptide bond and many amino acid molecules form a polypeptide (Johnson & Miller 1997). Most amino acids are present as two active isomers which are D (dextrorotary) and L (levorotary) forms. These forms are called enantiomers which are mirror images of each other (Robins et al. 2001). Amino acids are synthesis in the L-forms, but during life amino acid convert into mixture of the D- and L-forms. Thus, D-form of amino acids which accumulates during life has been related to aging process known as racemization (Bada 1982; McCudden & Kraus 2006). The degree of racemization of amino acid may be used for dating various fossil materials such as fossil shell (Hare & Abelson 1968), marine sediment (Kvenvolden 1973; Wehmiller & Hare 1971) and fossil bone (Bada 1985; Bada & Protsch 1973). In a previous study about amino acid in fossil reported that D-amino acid is derived from the change of L-amino acids in proteins. It was found the amount of racemization increased with age of the fossil and this is the first application of amino acid racemization in date events on the geological time scale (Bada & Protsch 1973).
RACEMIZATION OF ASPARTIC ACID
Aspartic acid is one of the non-essential amino acids group in the organism that can be formed in the human. Moreover, this amino acid is the component of some nutrition which is taken into the body. Since aspartic acid has fastest rate of racemization among all amino acids, it is used for an estimator in age determination. Aspartic acid racemization is non-enzymatic reaction within stable proteins or long-lived proteins. There is accumulation of D-aspartic acids increased with age in human tissues and positive correlation was found between age and ratio of D/L aspartic acid. Thus, racemization of aspartic acid can be utilized for age indicator of individuals and also investigated protein aging. In addition, there was found that aspartic acid racemization process involve some pathological diseases (Bada et al. 1973; Jousse et al. 2004; Ritz-Timme & Collins 2002).
Pathway of aspartic acid racemization occurs in proteins via a succinimide intermediated (succinyl residue or Asu) and the chiral center of succinimide can be easily racemized. Succinimide intermediate can be formed from L-aspartic acid which converts into D-aspartic acid later (Robins et al. 2001). Succinimide intermediate is formed by nucleophilic attack process that begin from the nitrogen of the C-terminal peptide bond attract to the carbon atom of carboxyl group resulting in cyclization (connection together in the form of ring). The succinimide ring is sensitive to racemization due to their instability so racemization rapidly occurs in succinimide ring. It make this structure can be converted from L- form of aspartic acid to D-form of aspartic acid (McCudden & Kraus 2006). Therefore, rate of racemization depends on the structure of amino acids within polypeptide that related to rapid accumulation of D-aspartic acids in proteins (Geiger & Clarke 1987; Radkiewicz et al. 1996; Ritz-Timme & Collins 2002).
FACTORS TO RACEMIZATION
The previous study investigated amino acid racemization from dental enamel in human and the results showed that there was different degree of racemization between different types of teeth. Amino acid racemization in tooth molars is more rapid than tooth incisors. Different racemization may result from temperature. Since tooth molars are located at the back of the mouth, there is slightly higher temperature than other teeth which have cooler temperature (Ohtani et al. 2003). This indicates that the higher the temperature, faster is the racemization rate. In the preparation of sample before analysis step, the sample should be stored at low temperature (McCudden & Kraus 2006; Ohtani et al. 2005). There are studies on the influence of pH for aspartic acid racemization of age estimation in dentin. The researchers stored the teeth in many solutions with different pH condition consisting of acidic (pH 4), alkaline (pH 9) and distilled water, and dry conditions. It was found that the rate of racemization or the reaction rate constant was highest in a pH 9 solution, then distilled water, in pH 4 solution, and in dry condition, respectively. The estimated age from tooth dentin in different environments for 1 year showed that there was slight increase by 0.007 years in a dry condition, 0.1 years in pH 4 condition, and 0.2 in water. On the other hand, teeth dentin left in the pH 9 solution found that the estimated age increased by 0.6 years. Moreover, the teeth left in the pH 9 solution for 5 years showed increase of 3.2 years. It is suggested that in alkaline condition may be more influent to amino acid racemization than other conditions (Ohtani 1995a). Moreover, fixation of sample is one of important factors to amino acid racemization. Ohtani and colleagues studied age estimation from human teeth by using amino acid racemization related influence of fixative. They used the teeth from cadavers and stored teeth in different fixatives include 95% ethanol, 10% formalin solution or 10% neutral formalin fixative at different temperatures. The results showed rate of aspartic acid racemization was highest in 10% neutral formalin solution, then in 10% formalin solution, and in 95% ethanol, respectively. In addition, it was found that the teeth stored at 15