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Aspartic Acid Racemization with Correlation to Age: AForensic Perspective

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ORIGINAL ARTICLE

Aspartic Acid Racemization with Correlation to Age:

A Forensic Perspective
Qudsia Hassan1, Allah Rakha2 and M. Zahid Bashir3

ABSTRACT
Objective: To estimate age through aspartic acid racemization of human dentin for forensic analysis, and to calculate the
margin of error between chronological age and estimated age by racemization method.
Study Design: Cross-sectional comparative study.
Place and Duration of Study: Dental Outpatients' Department, Ziauddin Hospital, Karachi, from 2011 to 2014.
Methodology: Patients from dental orthodontics department were selected. Verbal expressed consent was taken from the
participants. Preliminary data was entered in a specially designed performa having name, chronological age, gender,
socio-economic status. L and D ratio of aspartic acid were quantified in dentin for each tooth by High-Pressure Liquid
Chromatography technique. Results of 100 samples with known age were used during study to estimate co-efficient of
racemization. Regression equation was derived by plotting 85 samples of known age and KR as independent and
dependent variables and least square equation was derived - Age = -4.391 +347.396 (co-efficient of racemization).
Results: The estimated co-efficient of determination was 0.74, meaning thereby that the regression equation was 74%
correct for estimating age. The calculated correlation between chronological age and co-efficient of racemization was
0.834, which is interpreted as a very strong and positive correlation. The co-efficient of racemization increased with the
age. The mean chronological age (38.44 ±13.22) years were statistically similar to the mean calibrated age (37.52 ±12.36)
years. The median of actual and calibrated age was found to be 3.4 with inter-quantile value of 13. No statistically
significant difference was observed among gender (p=0.837), or socioeconomic status (p=0.35).
Conclusion: Racemization of human dentin could be used as one of the reliable method for estimating age. HPLC is a
reliable technique to estimate the co-efficient of racemization because it is highly reproducible, cost-effective and
convenient. However, the procedure should be optimized and standardized within all laboratories for quality assurance.

Key Words: Aspartic acid. Dentin. HPLC. Co-efficient of racemization (KR). Age estimation.

INTRODUCTION printing methods.1 In dead persons and archaeological

Age estimation has always been a sensitive issue in
Forensic Medicine as it is an essential requirement in
living individuals as well as in dead, cadavers, and
human remains. In the living individual, the importance
of age estimation is required from school going to
marriage, from employment to getting civil rights, and
criminal proceedings. Similarly, age estimation is an
important parameter in personal identification of
cadavers and human remains due to high rise in
terrorism in a country like Pakistan. The protocol in
identification of an unknown body comprises of
determination of gender, age, race and stature,
respectively. Gender is determined by DNA or finger
1 Department of Forensic Medicine and Toxicology, Ziauddin
University, Karachi.
2 Department of Forensic Sciences, Quality Assurance Cell,
University of Health Sciences, Lahore.
3 Department of Forensic Medicine, Shalamar Medical and
Dental College, Lahore.
Correspondence: Prof. Qudsia Hassan, Professor and Head
of Department, Department of Forensic Medicine and Toxicology,
Ziauddin University, Karachi.
E-mail: drqudsiahassan@hotmail.com
Received: November 04, 2016; Accepted: June 01, 2017.
samples of bones, assessment of age is one of the
foremost parameters to build mortality profile of the dead
and is of utmost forensic importance. Therefore, these
estimations should be as accurate as possible.2
The standard protocol to assess age in living and dead
is based on naked eye changes and radiological
examination of skeletal and dental tissues. In skeletal
tissues, fusion of bones and fusion of skull sutures are
done; whereas, eruption timings and wear and tear of
various teeth are assessed. The standard protocol to
assess age from teeth in children and adolescents are
based on measuring length of crown and root of teeth by
radiological method, eruption timeframe of deciduous
and permanent teeth, calculating mineralization level of
teeth, and measuring extent of wear and tear of
permanent teeth known as Gustafson's method.3 In
living adults and in dead, especially in cases of mass
disasters, morphological and histological method of age
assessment are much less accurate. Age estimation
protocols, based on morphological methods, are not
accurate and give error margins of greater than > ±10
years. Biochemical methods, which include aspartic acid
racemization of dentin, are one of the precise methods
of forensic age estimation with error margin of 3 ±1 year.
At present, aspartic acid racemization of dentin,

Journal of the College of Physicians and Surgeons Pakistan 2017, Vol. 27 (5): 283-287 283
Qudsia Hassan, Allah Rakha and M. Zahid Bashir
combined with radioactive carbon dating of teeth
enamel, represents method of choice for precise age
estimation.1
L-amino acid are transformed into D-amino acid by
racemization, which gradually accumulates in tissues
during the course of aging. This conversion is more
significant in tissues where metabolism precedes slowly
like bradytropic tissue, i.e. bone, eye lens, white matter
of brain and teeth.3 Among the amino acids, aspartic
acid has the fastest racemization rate. Amino acids
structurally bind to form peptides and proteins in L-form
at the time of birth; and with time, it starts transforming
into D-form. So, D and L forms, of amino acids exist in
a racemic mixture in every tissue, provided there is no
subjection to physiological renewal. In stable tissues like
dentin the rate of conversion is 0.1% per year.4
D-aspartic acid increases correspondingly with age in
dentin of human teeth; and hence, a good indicator of
age.5-8 Helfman and Bada were first researchers who
found that correlation between D and L aspartic acid with
age is highest r = 0.979 in dentin crown.
The correlation of dentin with chronological age in an
individual by aspartic acid racemization has been
demonstrated in a large number of studies.5-8 For
Pakistani population, results of a pilot study on 8
samples were found to be consistent with previous
studies.9
The objectives of this study were to calculate age by
aspartic acid racemization in human dentin, and
determine the margin of error between chronological
and estimated age by racemization method.
METHODOLOGY
This study was conducted in dental outpatients' clinics at
Ziauddin Hospital, Karachi, from 2011 to 2014. It was a
cross-sectional study and sampling technique was
convenient sampling. The sample size was calculated
through open Epi based on 50% proportion, 95%
confidence level and bound of error of 10%, using the
formula n = Z P2(1-P). The computed sample size would be
2
d
n = 97 for the given study. One hundred samples were
processed finally for analysis.
Healthy teeth, which were taken out during various
orthodontic procedures, were utilised for this research
including incisors, canines, premolars, and 1st and 2nd
molars. Third molars and filled and carious teeth were
being excluded from study.10
Informed verbal consent was taken from patients who
agreed to participate in this study. Performa was
specially designed for this study. The data included in
the performa was name, age, gender and socio-
economic status. Approval was taken from the
Institutional Review Board (IRB) of Ziauddin University.
284 Journal of the College of Physicians and Surgeons Pakistan 2017, Vol. 27 (5): 283-287
One hundred samples of teeth were collected and
stored. HPLC was performed after these samples were
demineralised, hydrolyzed, and derivatized in dentin.10-12
SPSS version 20 was utilized for data entry and
computation was done as well. Data was comprised on
quantitative and qualitative information of patients since
frequencies and percentages were used to express
qualitative variables, i.e. gender and socio-economic
status. Pearson Correlation was applied to find
correlation between age and co-efficient of racemization,
and p-value ≤ 0.05 was taken as significant. Data was
depicted in a graph by a scatter plot. Eighty-five samples
were employed for linear regression analysis to find the
relationship between actual age and the degree of
racemization. Linear regression equation was derived to
determine the predicted value of age of remaining 15
samples. Co-efficient of determination (r2) was used to
measure the accuracy of regression equation. Paired
t-test was employed to find the error between chrono-
logical ages and calibrated ages. Comparative analysis
of actual and calibrated age was represented by multiple
line charts in same graph. Independent sample t-test
was applied to find out the difference between co-
efficient of racemization (KR) and gender by considering
level of significance ≤ 0.05. Difference between co-
efficient of racemization (KR) and age categories, and
socio-economic status were tested out by one-way
ANOVA after the validity check of normality test, (level of
significance ≤ 0.05). The regression line equation
derived from least square method was represented as
age = -4.391 +347.396 (co-efficient of racemization).
RESULTS
There was very strong positive correlation of 0.834 with
p < 0.0001 as seen in chronological age and co-efficient
of racemization in 85 samples. This can be interpreted
as: with increase in age progressively, co-efficient of
racemization is increased (Figure 1). Results of 85
Figure 1: Correlation between age (years) and Co-efficient of Racemization
(KR).
Aspartic acid racemization with correlation to age

samples were used during study to estimate age (Table I). Table II: Calibrated age by regression formula and comparison with
Regression equation for estimating age was 74% actual age.
correct, meaning thereby that value of co-efficient of Code Actual age* KR Calibrated age Error
determination was 0.74. Fifteen samples were treated Unknown 01 23 .05 24.50 1.50

as unknown age group samples. Ages of these 15 Unknown 02 32 .09 27.99 4.01

samples were calculated by regression equation derived Unknown 03 34 .04 40.25 5.75

above, to check whether computed regression line is

Unknown 04 37 .08 31.25 6.75
Unknown 05 37 .11 35.02 1.98
good estimator to predicted age (Table II). The estimated
Unknown 05 39 .09 31 8.01
age of 15 samples showed that difference between Unknown 06 40 .10 30.35 9.65
mean actual age (38.44 ±13.22 years) and mean Unknown 07 43 .11 33.82 9.18
calibrated age (37.52 ±12.36 years) was not statistically Unknown 08 42 .15 47.72 -5.72
different (p=0.222). The median of actual and calibrated Unknown 09 45 .16 51.19 -6.19
age was found to be 3.4 with interquantile value of 13. Unknown 10 52 .16 51.19 .81
Unknown 11 55 .17 54.67 .33
Table I: Information regarding 85 samples KR (co-efficient of Unknown 12 57 .17 54.67 2.33
racemization) = ln (1+D/L)/(1-D/L).
Unknown 13 60 .18 58.14 1.86
Sample Actual Age KR Sample Actual Age KR
Unknown 14 61 .18 58.14 2.86
1 12 0.06 44 37 1.15
Unknown 15 16 .05 14.37 1.73
2 15 0.06 45 37 1.12
Age = -4.391 +347.396 (co-efficient of Racemization) is used for calculating calibrated age.
3 16 0.07 46 37 1.11 KR (co-efficient of racemization) = ln (1+D/L)/(1-D/L), Error = (Actual age - Calibrated age).
4 18 0.06 47 37 1.14
5 19 0.07 48 37 1.12 Table III: Comparision among co-efficient of racemization among race,
6 20 0.08 49 37 1.13 socio-economic and age categories (post-hoc test).
7 20 0.08 50 38 1.15 Variables Co-efficient of racemization (KR) P-value
8 21 0.09 51 38 1.15 Mean ± SD
9 22 0.09 52 38 1.13 Gender
10 22 0.09 53 39 1.14 Male (62) 0.120±0.035 0.837
11 22 0.11 54 39 1.13 Female (38) 0.121±0.0356
12 25 0.11 55 40 1.11 Socio-economic status
13 25 0.11 56 40 1.11 Low 0.127 ± 0.0414 0.358
14 25 0.1 57 40 1.12 High 0.115 ± 0.031
15 25 0.1 58 40 1.13 Medium 0.114 ± 0.035
16 25 0.11 59 42 1.15 Age category (years)
17 25 0.12 60 42 1.16 12 -22 0.0862±0.008 <0.0001
18 25 0.11 61 42 1.15 23 - 33 0.097 ± 0.021
19 26 0.11 62 42 1.14 34 - 44 0.113 ±0.025
20 26 0.1 63 43 1.14 45 -55 0.158 ± 0.008
21 26 0.12 64 43 1.15 Above 55 0.173 ±0.0084
22 27 0.12 65 43 1.13
23 27 0.11 66 45 1.15 There was no statistically significant difference observed
24 28 0.13 67 45 1.12 among gender (p=0.837, Table III). Socio-economic
25 28 0.12 68 47 1.15 status also did not show significant difference among the
26 28 0.05 69 47 1.14 mean values of KR, i.e. p=0.35 (Table III). In contrast,
27 30 1.12 70 49 1.15 mean values of KR were significantly different among
28 30 1.11 71 52 1.15
different age categories (p<0.0001). To explore further
29 30 1.16 72 52 1.16
significance between each group for KR considering age
30 30 1.17 73 55 1.15
categories, post hoc tests named as LSD were applied
31 31 1.18 74 55 1.15
32 31 1.17 75 56 1.15
(Table II). The mean value of KR, when age group of
33 32 1.11 76 57 1.16
12-22 years was compared with 22-33 years, was not
34 32 1.13 77 57 1.16 statistically significant (p= 0.124). Significant differences
35 32 1.11 78 59 1.16 were observed when 12-22 years age group was
36 32 0.099 79 60 1.17 compared in the 34-44 years (p< 0.001), 45-55 years
37 34 1.09 80 61 1.17 (p < 0.001), and above 55 years (p < 0.001). Similarly,
38 34 1.09 81 61 1.17 significant difference was observed when 23-33 years
39 35 1.13 82 63 1.18 age group was compared with 34 - 44 years (p = 0.002),
40 35 1.33 83 65 1.18 45-55 years (p < 0.001), and 55-years above (p < 0.001).
41 35 1.14 84 70 1.18 Borderline significant differences were observed when
42 36 1.14 85 70 1.19 45-55 years age group was compared with 55-years
43 37 1.15
and above group (p= 0.051).

Journal of the College of Physicians and Surgeons Pakistan 2017, Vol. 27 (5): 283-287 285
Qudsia Hassan, Allah Rakha and M. Zahid Bashir
DISCUSSION
Teeth are one of the hardest and most resistant tissues
in human body, which make it so important in forensic
analysis. Teeth can be used in identification even after
all other tissues, especially soft tissues, have
disintegrated; and if antemortem dental records are
available, these postmortem findings can be matched.1
Dentin is considered as ideal tissue for forensic age
analysis.13 Aspartic acid racemization analysis of the
human dentin determines the chronological age of the
individual at the time of death as conversion of L-form
ceases to D-form at the time of death.1,2
The results of the present research showed a strong
correlation (0.834) between chronological age and co-
efficient of racemization (Figure 1). The results of this
research also revealed that calculated co-efficient of
determination, difference between mean of actual age
and mean of calibrated age, and the median actual and
calibrated age, were validated by other studies that
explored aspartic acid racemization on tooth dentin.2
Researches were conducted on unerupted and
supernumerary teeth, and on enamel, cementum and
dentin, which obtained similar results.14-16 Further
researches conducted on dentin and enamel on same
teeth of same individual reported to have a higher
correlation with age in dentin than enamel.1,5 In other
studies, other amino acids like alanine and glutamate
were employed to calculate degree of racemization; but
most accurate results were achieved from aspartic acid
racemization.6,17-19
Same level of accuracy is not achieved in all researches
and inconsistencies do exist.19 However, aspartic acid
racemization age analysis of dentin allows for a fair
prediction of age of the person with a precision as
reported in previous studies, and which is also in
accordance to the results of this study.2,20 Several
factors influence the process of racemization like
ambient temperature, PH, and humidity. Teeth are
exposed to different ambient temperatures depending
on their locations.1 The influence of ambient
temperature will be stronger than period of tooth
formation in oral cavity as seen in elderly.3
Similarly, bacterial infections like staphylococci affect
racemization. Jir et al. made a study using non-
collageneous protein from dentin for assessment of age
where staphylococcal proteaseV8, specifically splits
peptide bonds in L-glutamic acid or L-aspartic acid.21
Fire enhances the conversion rate from L-form to D-form
so the racemization rate will be higher in burned bodies
and can be a source of error.22
Previous data concluded that use of the same type of
tooth throughout would reduce errors in estimation of the
exact age of dentin, since it eliminates systematic
variation due to different developmental rates for
different teeth. This difficulty was overcome by
substituting age specific standard specimens.23,24
286 Journal of the College of Physicians and Surgeons Pakistan 2017, Vol. 27 (5): 283-287
Similarly, differences of methodologies induce differences
in racemization rates; and hence, affect age estimation.
GC and HPLC both had been used but HPLC have
proven to be a better technique in terms of
reproducibility, higher sensitivity, and shorter analysis
time. Recently derivatization with OPA with NAC is the
method of choice, allowing achievement of good
selectivity and good resolution.11,12,25 Small quantities of
amino acids as low as 0.9 pg can be employed in
reverse phase-high pressure liquid chromatography
(RP-HPLC) with fluorescence detection.11,25
When post-hoc tests named least square difference
(LSD) was applied to different age categories, it was
found that racemization rates differ in young and middle
age versus elderly individuals. This result is in
accordance to previous researches conducted on
middle and elderly age groups. This can be interpreted
by the fact that in elderly individuals ambient
temperature plays a vital role on racemization process
than the time of tooth formation.1,3 Similarly, degree of
racemization in dentin among different types of teeth
from same individual are different according to time of
tooth formation in middle aged people, but not in elderly
people.3 The LSD result applied to different age
categories can be interpreted in terms: as age
increases, the error of margin forage estimation
increases due to changes in the internal environment of
teeth.
Standard recommendations should be outlined in
forensic science laboratories for comparability and
reliability of results.2 Use of the same type of tooth
throughout would reduce errors in estimation of the
exact age of dentin, since it eliminates systematic
variation due to different developmental rates for
different teeth. Age specific standard specimens can be
substituted so racemization ratio can be measured with
sufficient reproducibility. Ideally, HPLC should be
coupled with radioactive carbon dating for precise and
accurate age estimations.1
CONCLUSION
Aspartic acid racemization is an accurate method for
calculating age in living as well as in dead individuals.
The mean actual age (38.44 ±13.22 years) was not
significantly different from the mean calibrated age
(37.52 ±12.36 years). No statistically significant
difference was observed among gender or socio-
economic status. HPLC remains the method of choice in
forensic age estimations with error of margin ±3 years.
Acknowledgment: This research is financially
supported and funded by Higher Education Commission
of Pakistan by project titled “Amino acid racemization
in human dentin as an indicator of age project No. 20-
1759/R&D/10. Final report of project was submitted in
august 2014. The support provided by Higher Education
Commission is greatly acknowledged.
Aspartic acid racemization with correlation to age
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