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Estimation of Postmortem Interval Using Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy in Adipose Tissues
Estimation of Postmortem Interval Using Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy in Adipose Tissues
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Original Article
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
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
[Downloaded free from http://www.jfsmonline.com on Tuesday, December 21, 2021, IP: 182.253.124.72]
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
high‑throughput data, and its application in different fields to caused by asymmetric C–H stretching (=C–H). The peak
address a variety of problems has increased in recent years.[23] at 1743 cm−1 is due to the C =O stretching mode of lipids.
The PLS attempts to find factor latent variables (LVs) that Compared with the spectrum at 0 days, the spectrum at
maximize the amount of variation explained in X that is 6 days showed a new peak at 1711 cm−1, which is known as
relevant for predicting response Y and modeling the linear a significant region for free fatty acids. We considered that
relationship between X and Y, such as the spectral data and this may be a characteristic peak related to PMI. The peak at
the PMI values in this study. A critical step in optimizing the 1465 cm−1 is assigned to the CH2 scissoring mode of the acyl
predictive ability of the model is to determinate LVs, which chain of lipids, and the frequency range of 1159–1174 cm−1
usually depends on cross‑validation (CV). In this case, we results from stretching vibrations of carbohydrates. The
adopted leave‑one‑out CV, and the selection of an optimal spectral region between 1120 and 1050 cm−1 has two weak
number of LVs was based on the inclusion of additional factors peaks from RNA caused by C–O stretching at 1117 cm−1 and
only when the root mean square error of CV (RMSECV) PO2− symmetric stretching at 1089 cm−1.
improved by at least 5%. The value of the model is evaluated
The time of the peak at 1711 cm − 1 first appearing (T1711) in
by RMSECV, root mean square error of prediction (RMSEP),
eight samples during the 2 weeks is listed in Table 1. We
determination coefficient of CV (Rcv2), and determination
found that T1711 of sample 1 and sample 7 are 6 and 8 days,
coefficient of prediction (Rp2). RMSECV generally indicates
respectively, which are far less than those of other samples, and
the magnitude of global model error in the calibration
a common characteristic of both is that they are in a state of high
model. RMSEP was used to evaluate the performance of the
putrefaction with longer PMI. Unfortunately, the PMI in both
prediction set in the prediction process. Lower RMSECV
cases cannot be fully determined due to the lack of on‑the‑spot
and RMSEP values, but small differences between these two
information. Excluding the two highly decomposed cases, the
values, indicate better predictive quality. The coefficient of
T1711 of sample 2 and sample 6 are both 10 days, which are
determination (R2) can evaluate the goodness of fit between
less than that in other samples. The PMI of these two samples
actual and predicted values, and the closer it is to one, the
is longer than that of the other samples. This finding further
better the model fits. MATLAB R2017b (The MathWorks,
confirmed the relevance of the peak at 1711 cm−1 to PMI.
MA, USA) equipped with PLS_Toolbox 8.6 (Eigenvector
Research, Inc., 3905 West Eaglerock Drive, Wenatchee, USA) Studies have proved that the body’s adipose tissue is mostly
was used for data analysis. composed of lipids, of which 90%–99% are triglycerides that
are hydrolyzed by intrinsic tissue lipases shortly after death to
Results and Discussion produce a mixture of saturated and unsaturated fatty acids.[21]
In the study by Swann et al., fatty acids could be detected
Visual spectral analysis of human samples by a method of gas chromatography–mass spectrometry in
Figure 1 shows the SNV‑pretreated ATR–FTIR spectra of fluids released during decomposition, and the components
sample 1 at 0 and 6 days in the range of 4000–400 cm−1. The of fatty acids were correlated with the PMI.[26] We found
assignment of the bands is shown in Table 2.[24,25] The spectra that biomarkers (free fatty acids) can also be detected by
have strong C–H absorption between 3050 and 2850 cm−1. ATR–FTIR in adipose tissue. The appearance of free fatty
The separate bands at 2922 and 2852 cm−1 correspond to acids resulting from the decomposition of lipids is correlated
asymmetrical C–H stretching (CH2) and symmetrical C–H with the PMI of the remains according to a preliminary study
stretching (CH2), respectively, with a weak peak at 3008 cm−1 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
to verify the findings on human samples and to establish a related to the amide II region, informed us about the mixing
suitable model to estimate PMI. of short peptide chains.
Mouse sample spectral analysis In the next step, we applied chemometric methods to elucidate
We used only the spectral regions at 3050–2800 cm−1 and more information from the spectral dataset to construct a model
1800–400 cm−1, which contained the fundamental vibrational for PMI estimation. Figure 3 shows the relatively satisfactory
energy‑absorbing frequencies of most biomolecules for further result (R cv2 = 0.80, R p2 = 0.80; RMSECV = 1.78 days,
analysis to reduce the interference from noise. Figure 2 shows RMSEP = 1.87 days) of the PLS model with 4 LVs in
the average spectra after pretreatment with SNV of every leave‑one‑out CV after data were preprocessed by the SNV
PMI group and their comparison. The assignments of the method. Furthermore, we calculated the variable importance
peaks are listed in Table 2 and described above. Compared in projection (VIP) scores for all spectral variables to account
to the uncontrollability and limitations of human samples, for this model.[27] Variables with VIP scores above 1.0 were
we can clearly determine the postmortem trends in mouse considered to be influential for model construction. The
adipose tissue. In Figure 2, the most conspicuously changed higher the VIP scores for each variable, the more important
peaks are at 1743 and 1711 cm−1, and the intensity of 1743 the variable to the PLS model. Figure 4 shows the highest
cm−1 decreased over time, while 1711 cm−1 increased at first peaks of VIP scores at 1743 and 1711 cm−1 which belonged
and then decreased. The peak associated with = C–H at to C=O vibrations of lipids and free fatty acids, followed by
3008 cm−1 displayed an increasing trend in intensity, while C–O vibrations from carbohydrates. Meanwhile, asymmetric
the peak related to CH2 vibration at 2922 and 2852 cm−1 stretching and scissoring of CH2 and amide II constitute less
displayed a trend of rising first and then falling. We believe influential variables. The VIP scores further reflect that the
that this change in the spectrum results from the hydrolysis of process of triglyceride degradation into fatty acids is relatively
triglycerides, which reduces the content of lipids and increases regular and has a stable trend, which is of great significance
the content of free fatty acids, and with an increasing PMI, free for estimating PMI.
fatty acids dispersed from the adipose tissue and became part
In vivo experiments in mice, we found that the postmortem
of the degradation products. Furthermore, we can determine
changes were consistent with that of human adipose tissue
that the time when the peak at 1711 cm−1 first appeared is on
in vitro after death. This finding is of great significance for
the 4th day at 25°C ± 1°C and a relative humidity of 50% ± 5%.
the investigation of the time of death of decomposed remains
The variation of the spectrum on the characteristic peak is
in the future. Comparing the degradation rule to other tissues,
consistent with the human sample in vitro. The difference is
changes in adipose tissue were mainly observed after 4 days,
that the characteristic peak in mouse samples in vivo appeared
while those in the liver and spleen were before 6 days, and
earlier than in human samples in vitro. We consider adipose
when the PMI goes to over 6 days, the tissues full of protein
tissue in vivo to be degraded in the same way as in vitro, but
can easily be influenced by microbes according to Huang’s
happening more quickly, due to the richer content of enzymes
finding.[19] In addition, Wang et al. collected spectral data of
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
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