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Original Article

Estimation of Postmortem Interval Using Attenuated Total

Reflectance: Fourier Transform Infrared Spectroscopy in
Adipose Tissues
Haohui Zhang1, Qi Wang2, Kai Zhang1, Ruina Liu1, Shuanliang Fan1, Zhenyuan Wang1
1
Department of Forensic Pathology, Xi’an Jiaotong University School of Medicine, Xi’an, China, 2Department of Forensic Medicine, Chongqing Medical University,
Chongqing, China

Abstract
Estimation of postmortem interval (PMI) is vitally important in forensic investigations. Although many studies have examined the chemical
changes of various tissues over time, no reports using spectroscopic methods in adipose tissue are available. In this study, attenuated total
reflectance–Fourier transform infrared (ATR–FTIR) spectroscopy was utilized to collect comprehensive biochemical information from human
adipose tissues in vitro at different times. Thereafter, mice were used as samples for in vivo experiments for more detailed studies on PMI. Then,
partial least squares (PLS) model for PMI estimation was established based on the acquired spectral dataset of mouse samples. The spectral
variable associated with C=O arising from lipids and free fatty acids was most susceptible to PMI. Moreover, the PLS model appeared to achieve
a satisfactory prediction with a root mean square error of cross‑validation of 1.78 days, and the reliability of the model was determined in an
external validation set with a root mean square error of prediction of 1.87 days. The study shows the possibility of application of ATR–FTIR
methods in PMI estimation using adipose tissue.

Keywords: Adipose tissue, attenuated total reflectance–Fourier transform infrared, chemometrics, postmortem interval

Introduction Fourier transform infrared (FTIR) spectroscopy is a highly

sensitive analytical method for identifying the molecular
Estimation of postmortem interval (PMI), also known as time
composition of biological samples according to the detection
since death, is vitally important in forensic investigations.
of vibrational mode of their chemical bonds. Because of its
In daily forensic casework, a precise estimation of the PMI
nondestructive, rapid, portable, and user‑friendly advantages,
can help forensic pathologists verify the witness statement,
FTIR has been widely used in forensic investigations to detect
narrow the search scope, or even guide the direction of
and analyze fingerprints, inks, fibers, hair, and gunshot residues
the investigation. Various methods have been explored
found at crime scenes.[8‑12] In addition, FTIR can be applied to
by forensic scholars to achieve this purpose.[1] Traditional
analyze biological samples, including organic macromolecule
methods include the examination of algor mortis, rigor
components, such as proteins, carbohydrates, lipids, and
mortis, livor mortis, and the growth state of insects after
nucleic acids.[13] As organs and tissues degrade gradually after
death. [2,3] Many new methods focus on the postmortem
death, the components of the tissues on the remains change
changes in chemical and biomolecule indices, such as the
over time, and this phenomenon can be reflected in the spectral
degradation of DNA, RNA, protein, and the variation of
postmortem microorganisms.[4‑7] However, most of these
new methods require complex processes to analyze forensic Address for correspondence: Dr. Zhenyuan Wang,
Department of Forensic Pathology, Xi’an Jiaotong University
samples, and the instruments required are expensive and School of Medicine, Xi’an, China.
time‑consuming. Therefore, the use of a convenient and E‑mail: wzy218@xjtu.edu.cn
simple pretreatment for estimating the PMI would be quite
beneficial in forensic daily work.
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How to cite this article: Zhang H, Wang Q, Zhang K, Liu R, Fan S, Wang Z.
DOI: Estimation of postmortem interval using attenuated total reflectance: Fourier
10.4103/jfsm.jfsm_47_18 transform infrared spectroscopy in adipose tissues. J Forensic Sci Med
2019;5:7-12.

© 2019 Journal of Forensic Science and Medicine | Published by Wolters Kluwer - Medknow 7
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Zhang, et al.: PMI estimation using ATR-FTIR in adipose tissues

data, such as the decline in the peak intensities and areas. In constant temperature of 25°C ± 1°C and a relative humidity
combination with chemometrics, more spectral information can of 50% ± 5%. Then, approximately 0.5 cm × 0.5 cm × 0.5 cm
be obtained and more appropriate models can be established of adipose tissue was sheared from each sample every 2 days
to estimate PMI.[14,15] for the next 2 weeks. To obtain stable spectral images,
pretreatment was performed before sample measurement: all
Previous works by the authors have demonstrated the
small tubes of the adipose tissues were ultrasonically ground
usefulness of FTIR for the study of biochemical changes
for 15 s and centrifuged at 4°C and 3000 rpm for 3 min, and
in organs and biological fluids, and several characteristic
the supernatants were obtained and then kept frozen at −80°C
peak intensities and areas were proved to be correlated with
until FTIR analysis.
PMI.[1,16‑19] Considering that most tissues are susceptible
to degradation and microbial interference, there are many Animal sample preparation
limitations to accurately estimating PMI for remains in a state Male KM mice (n = 121, weight 24–26 g), purchased from the
of high putrefaction. Yan et al. found that the composition of Animal Center of Xi’an Jiaotong University, were anesthetized
adipocere is different at various points of time, which can be by 0.1 ml/10 g 4% chloral hydrate through the abdomen, and
applied to estimate PMI in specific cases.[20] The degradation were then sacrificed by cervical dislocation. No sample was
of lipids is relatively slow and can be applied for longer PMI collected prior to sacrifice. All the animal experiments in
estimation.[21] Moreover, adipose tissue is close to the body the present study were specifically approved and overseen
surface and can be obtained through a small incision, even by the Care and Use of Laboratory Animal Committee of
without performing a forensic autopsy, and it may be an Xi’an Jiaotong University. Cadavers were kept at moderate
appropriate sample to estimate PMI of highly decomposed ambient temperatures of 25°C ± 1°C and a relative humidity of
remains. Thus, in this work, we employed FTIR coupled with 50% ± 5% in an environmentally controlled chamber following
chemometrics to detect the decomposition process of adipose sacrifice. Adipose tissue samples from 121 mice were taken
tissue. at 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, and 14 days (11 samples for each
time point, eight for a calibration set, and three for a prediction
Materials and Methods set). All adipose tissue samples were collected from the
inguinal region. Because the mice did not have enough lipid
Human sample preparation
in their adipose tissue, all samples were ultrasonically ground
Informed consent was obtained from the relatives of all
for 15 s, and 100‑μL petroleum ether was added to purify the
deceased individuals in this study, and it must be emphasized
lipids in adipose tissue. Moreover, it must be noted that the
that all procedures in this study comply with the requirements
effumability of petroleum ether should not impact the spectral
of local laws and institutional guidelines and were approved
data collection. Then, the samples were centrifuged at 4°C
and overseen by the Ethics Committee of Xi’an Jiaotong
and 3000 rpm for 3 min, and the supernatants were mainly
University. A total of eight subcutaneous adipose tissues of
contained in adipose tissue for FTIR analysis.
the abdomen were collected from eight human remains: two
were highly decomposed and six were relatively fresh, as Fourier transform infrared measurements
shown in Table 1. For each case, after a longitudinal incision FTIR spectra were collected by a Thermo Nicolet–IS50
of the whole abdominal wall during the autopsy, approximately FTIR spectrometer (Thermo Electron Scientific Instruments
4 cm × 4 cm of skin with the full layer of subcutaneous fat Corp., WI, USA) coupled with a diamond‑attenuated total
was cut off from above the umbilical area. Then, the collected reflectance (ATR) accessory, and a KBr beam splitter was used
specimens were preserved in a 100‑mL measuring cup and for spectral acquisition. The infrared spectra analysis software
placed in an environmentally controlled chamber with a package OMNIC version 8.2 (Thermo Nicolet Analytical
Instruments, WI, USA) was used for analyzing the spectra and
recording the data. Three 1‑μL supernatants of each sample
Table 1: Human sample information
were added drop wise to the diamond ATR crystal. To ensure
n Age Gender Sign of PMI (day) T1711* (day) spectral reproducibility and assess analytical precision, every
(year) putrefaction drop of the supernatant was detected and recorded three times.
1 23 Male Bloated cadaver, ‑ 6 The parameters of spectral collection were set to frequencies
putrefactive blister
ranging from 4000 to 400 cm−1 with a resolution of 4 cm−1
2 33 Female Fresh 1–2 10
3 41 Male Fresh <1 >14
and 32 scans.
4 46 Male Fresh <1 >14 Attenuated total reflectance–Fourier transform infrared
5 34 Female Fresh <1 >14
spectral pretreatment and analysis
6 47 Male Fresh 1–2 10
For each sample, nine replicate spectra were obtained,
7 28 Female Bloated cadaver, ‑ 8
putrefactive blister
and the mean spectrum was used for dataset treatment. To
8 64 Female Fresh <1 >14 reduce systematic variations, we used the standard normal
*T1711 means the time of the peak at 1711/cm first appearing. PMI: variate (SNV) method to normalize the spectra.[22] Partial
Postmortem interval least squares (PLS) is a popular modeling approach for

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Zhang, et al.: PMI estimation using ATR-FTIR in adipose tissues
high‑throughput data, and its application in different fields to
address a variety of problems has increased in recent years.[23]
The PLS attempts to find factor latent variables  (LVs) that
maximize the amount of variation explained in X that is
relevant for predicting response Y and modeling the linear
relationship between X and Y, such as the spectral data  and
the PMI values in this study. A critical step in optimizing the
predictive ability of the model is to determinate LVs, which
usually depends on cross‑validation (CV). In this case, we
adopted leave‑one‑out CV, and the selection of an optimal
number of LVs was based on the inclusion of additional factors
only when the root mean square error of CV (RMSECV)
improved by at least 5%. The value of the model is evaluated
by RMSECV, root mean square error of prediction (RMSEP),
determination coefficient of CV  (Rcv2), and determination
coefficient of prediction (Rp2). RMSECV generally indicates
the magnitude of global model error in the calibration
model. RMSEP was used to evaluate the performance of the
prediction set in the prediction process. Lower RMSECV
and RMSEP values, but small differences between these two
values, indicate better predictive quality. The coefficient of
determination (R2) can evaluate the goodness of fit between
actual and predicted values, and the closer it is to one, the
better the model fits. MATLAB R2017b  (The MathWorks,
MA, USA) equipped with PLS_Toolbox 8.6 (Eigenvector
Research, Inc., 3905 West Eaglerock Drive, Wenatchee, USA)
was used for data analysis.
Results and Discussion
Visual spectral analysis of human samples
Figure 1 shows the SNV‑pretreated ATR–FTIR spectra of
sample 1 at 0 and 6 days in the range of 4000–400 cm−1. The
assignment of the bands is shown in Table 2.[24,25] The spectra
have strong C–H absorption between 3050 and 2850 cm−1.
The separate bands at 2922 and 2852 cm−1 correspond to
asymmetrical C–H stretching (CH2) and symmetrical C–H
stretching (CH2), respectively, with a weak peak at 3008 cm−1
Figure 1: The standard normal variate pretreated attenuated total
reflectance–Fourier transform infrared spectra of sample 1 at 0 and 6 days
Journal of Forensic Science and Medicine  ¦  Volume 5  ¦  Issue 1  ¦  January-March 2019
caused by asymmetric C–H stretching (=C–H). The peak
at 1743 cm−1 is due to the C =O stretching mode of lipids.
Compared with the spectrum at 0 days, the spectrum at
6 days showed a new peak at 1711 cm−1, which is known as
a significant region for free fatty acids. We considered that
this may be a characteristic peak related to PMI. The peak at
1465 cm−1 is assigned to the CH2 scissoring mode of the acyl
chain of lipids, and the frequency range of 1159–1174 cm−1
results from stretching vibrations of carbohydrates. The
spectral region between 1120 and 1050 cm−1 has two weak
peaks from RNA caused by C–O stretching at 1117 cm−1 and
PO2− symmetric stretching at 1089 cm−1.
The time of the peak at 1711 cm − 1 first appearing (T1711) in
eight samples during the 2 weeks is listed in Table 1. We
found that T1711 of sample 1 and sample 7 are 6 and 8 days,
respectively, which are far less than those of other samples, and
a common characteristic of both is that they are in a state of high
putrefaction with longer PMI. Unfortunately, the PMI in both
cases cannot be fully determined due to the lack of on‑the‑spot
information. Excluding the two highly decomposed cases, the
T1711 of sample 2 and sample 6 are both 10 days, which are
less than that in other samples. The PMI of these two samples
is longer than that of the other samples. This finding further
confirmed the relevance of the peak at 1711 cm−1 to PMI.
Studies have proved that the body’s adipose tissue is mostly
composed of lipids, of which 90%–99% are triglycerides that
are hydrolyzed by intrinsic tissue lipases shortly after death to
produce a mixture of saturated and unsaturated fatty acids.[21]
In the study by Swann et al., fatty acids could be detected
by a method of gas chromatography–mass spectrometry in
fluids released during decomposition, and the components
of fatty acids were correlated with the PMI.[26] We found
that biomarkers (free fatty acids) can also be detected by
ATR–FTIR in adipose tissue. The appearance of free fatty
acids resulting from the decomposition of lipids is correlated
with the PMI of the remains according to a preliminary study
on human sample in vitro. However, the condition in vitro is
quite different from that in vivo. Due to the lack of continuous
research on lipid decomposition regularity, we studied mice
Table 2: Major band assignments of the Fourier transform
infrared spectrum of adipose tissue[24,25]
Wave number/cm Assignment
3008 Asymmetric C-H stretching(=C-H)
2922 Asymmetric C-H stretching (CH2)
2852 Symmetric C-H stretching (CH2)
1743 C=O stretching mode of lipids
1711 Free fatty acids
1545 Amide II
1465 CH2 scissoring
1159-1174 C-O stretching vibration of carbohydrates
1117 C-O stretching vibration of C-OH group of
RNA
1089 Stretching PO2‑symmetric in RNA
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Zhang, et al.: PMI estimation using ATR-FTIR in adipose tissues
to verify the findings on human samples and to establish a
suitable model to estimate PMI.
Mouse sample spectral analysis
We used only  the spectral regions at 3050–2800 cm−1 and
1800–400 cm−1, which contained the fundamental vibrational
energy‑absorbing frequencies of most biomolecules for further
analysis to reduce the interference from noise. Figure 2 shows
the average spectra after pretreatment with SNV of every
PMI group and their comparison. The assignments of the
peaks are listed in Table 2 and described above. Compared
to the uncontrollability and limitations of human samples,
we can clearly determine the postmortem trends in mouse
adipose tissue. In Figure 2, the most conspicuously changed
peaks are at 1743 and 1711 cm−1, and the intensity of 1743
cm−1 decreased over time, while 1711 cm−1 increased at first
and then decreased. The peak associated with = C–H at
3008 cm−1 displayed an increasing trend in intensity, while
the peak related to CH2 vibration at 2922 and 2852 cm−1
displayed a trend of rising first and then falling. We believe
that this change in the spectrum results from the hydrolysis of
triglycerides, which reduces the content of lipids and increases
the content of free fatty acids, and with an increasing PMI, free
fatty acids dispersed from the adipose tissue and became part
of the degradation products. Furthermore, we can determine
that the time when the peak at 1711 cm−1 first appeared is on
the 4th day at 25°C ± 1°C and a relative humidity of 50% ± 5%.
The variation of the spectrum on the characteristic peak is
consistent with the human sample in vitro. The difference is
that the characteristic peak in mouse samples in vivo appeared
earlier than in human samples in vitro. We consider adipose
tissue in vivo to be degraded in the same way as in vitro, but
happening more quickly, due to the richer content of enzymes
and water. The peak at 1159–1174 cm−1, which represents
carbohydrates, and the peaks related to RNA at 1117 and
1089 cm−1 decreased. This reduction in carbohydrates and
ribose is consistent with that of our previous research.[17,19]
The increased intensity of the peak at 1545 cm−1, which is
Figure 2: The average spectra after pretreatment with standard normal
variate of every postmortem interval group and their comparison
10 Journal of Forensic Science and Medicine  ¦  Volume 5  ¦  Issue 1  ¦  January-March 2019
related to the amide II region, informed us about the mixing
of short peptide chains.
In the next step, we applied chemometric methods to elucidate
more information from the spectral dataset to construct a model
for PMI estimation. Figure 3 shows the relatively satisfactory
result (R cv2 = 0.80, R p2 = 0.80; RMSECV = 1.78 days,
RMSEP = 1.87 days) of the PLS model with 4 LVs in
leave‑one‑out CV after data were preprocessed by the SNV
method. Furthermore, we calculated the variable importance
in projection (VIP) scores for all spectral variables to account
for this model.[27] Variables with VIP scores above 1.0 were
considered to be influential for model construction. The
higher the VIP scores for each variable, the more important
the variable to the PLS model. Figure 4 shows the highest
peaks of VIP scores at 1743 and 1711 cm−1 which belonged
to C=O vibrations of lipids and free fatty acids, followed by
C–O vibrations from carbohydrates. Meanwhile, asymmetric
stretching and scissoring of CH2 and amide II constitute less
influential variables. The VIP scores further reflect that the
process of triglyceride degradation into fatty acids is relatively
regular and has a stable trend, which is of great significance
for estimating PMI.
In vivo experiments in mice, we found that the postmortem
changes were consistent with that of human adipose tissue
in vitro after death. This finding is of great significance for
the investigation of the time of death of decomposed remains
in the future. Comparing the degradation rule to other tissues,
changes in adipose tissue were mainly observed after 4 days,
while those in the liver and spleen were before 6 days, and
when the PMI goes to over 6 days, the tissues full of protein
can easily be influenced by microbes according to Huang’s
finding.[19] In addition, Wang et al. collected spectral data of
Figure 3: The cross‑validation and prediction results of the partial least
squares model using spectral variables within 3050–2800 cm−1 and
1800–400 cm−1. The regression plot between the predicted and actual
postmortem interval. The red line represents the reference line of the
predicted PMI scores. The closer to it, the higher the goodness of fit
will be. The blue dot represents the calibration set, while the red square
represents the prediction set
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Zhang, et al.: PMI estimation using ATR-FTIR in adipose tissues
Figure 4: The plot of variable importance in projection scores displays
the contribution of the spectral variables to the distinction in the partial
least squares model. The variables with variable importance in projection
scores above 1.0 (marked by a red dot line) are considered most
significant
plasma and established models to estimate the PMI (within
2 days) with the application of chemometrics, which achieved
satisfying results (Rcv2 = 0.91, Rp2 = 0.85, RMSECV = 4.76 h,
RMSEP = 5.31 h), yielding a higher R2 than this study.[17]
We found that protein‑rich tissue had a better correlation
with the time of death in the short term, but had limitations
in inferring the PMI of degraded remains. In this study, we
found that adipose tissue compensates for this limitation.
As previous studies reported, some metabolites of lipids in
biological samples could be considered biomarkers for PMI
estimation.[20,26,28] However, they simply picked out possible
biomarkers and did not perform any subsequent research to
extrapolate the PMI. Based on their research, we established
a model to estimate the PMI by studying the consistent
postmortem changes of mice.
Along with the advantages of adipose tissue, the current
spectroscopic study suggests that adipose tissue may be
an appropriate medium for PMI estimation in a highly
decomposed body. However, the PMI is affected by many
factors, such as temperature, humidity, and cause of death,[29]
and the current study focused on a single condition, and the
application scope of the established model is limited. More
complex and changeable conditions in real forensic cases might
influence the process of postmortem changes of adipose tissues
and lead to larger prediction errors. Therefore, in future work,
we need to study the postmortem changes of adipose tissue
under complex conditions, establish corresponding models,
reduce errors, and adapt to the needs of real work.
Conclusion
In this work, ATR–FTIR spectroscopy was applied to acquire
biochemical information in adipose tissues of human samples
in vitro for the first time, and then in vivo tests were performed
on mice. The results show that the combination of FTIR
Journal of Forensic Science and Medicine  ¦  Volume 5  ¦  Issue 1  ¦  January-March 2019
analysis and chemometrics based on biochemical changes
in adipose tissue is ideal for estimating the PMI in highly
decomposed remains. Spectroscopic findings reflected that
PMI‑dependent changes in adipose tissue derived from the
time‑ordered process of hydrolysis of lipids into free fatty
acids, and other biological molecules, such as carbohydrates
and nucleic acids also have tiny effects. Moreover, the PLS
model was established to predict the PMI, which produced
satisfactory results. In summary, this study demonstrates the
feasibility of using ATR–FTIR on adipose tissue to estimate
the PMI and offers a promising new approach in the specific
scene of highly decomposed remains.
Financial support and sponsorship
This study was funded by the Council of the National Natural
Science Foundation of China (No. 81730056).
Conflicts of interest
There are no conflicts of interest.
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