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Estimation of post-mortem interval

using biochemical markers
a a
Andrea Evelyn Donaldson & Iain L Lamont
a
Biochemistry, University of Otago, 710 Cumberland Street,
Dunedin, 9016, New Zealand.
Published online: 29 Apr 2013.
To cite this article: Andrea Evelyn Donaldson & Iain L Lamont (2014) Estimation of post-mortem
interval using biochemical markers, Australian Journal of Forensic Sciences, 46:1, 8-26, DOI:
10.1080/00450618.2013.784356
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Australian Journal of Forensic Sciences, 2014
Vol. 46, No. 1, 826, http://dx.doi.org/10.1080/00450618.2013.784356
Estimation of post-mortem interval using biochemical markers

Andrea Evelyn Donaldson* and Iain L. Lamont
University of Otago, Biochemistry, 710 Cumberland Street, Dunedin, 9016, New Zealand
(Received 9 December 2012; nal version received 6 March 2013)
Determining the post-mortem interval (PMI) or time of death is a critical step in

Downloaded by [University of Winnipeg] at 11:15 11 September 2014
most homicide or un-witnessed death investigations. However, the various methods

currently used in estimating PMI yield large post-mortem windows and sometimes
contradict one another. Traditional biochemical methods have identied a number of
metabolites that change post mortem, but these have not been widely adapted for
determining PMI because they failed to produce precise, reliable, and rapid results
required by the forensic community. Recent developments in biochemical technolo-
gies are starting to identify biomarkers that may provide a much more accurate PMI
estimation. These techniques are more sensitive and specic than traditional methods
and can be standardised and statistically assessed, making them appealing methodol-
ogies. In this review, we discuss the use of biochemical methods for PMI determina-
tion in the early post-mortem period, with a particular focus on biochemical and
cellular changes occurring in the blood. We also provide an overview of current bio-
chemical methods and technologies that can be used to discover novel biomarkers
that may help to determine PMI.
Keywords: post-mortem interval; blood biomarkers; biochemical changes;
haematological changes
1. Introduction
The post-mortem interval (PMI) or time of death is a critical step in most homicide or
un-witnessed death investigations (including hospital deaths) and remains one of the
most challenging variables to quantify and establish1, despite the large amount of
research undertaken. Precisely determining PMI has deed detectives, medical examin-
ers and pathologists for over 2,000 years, with the earliest recorded rate of change
methodology being derived from the Egyptians and Greeks who described and pre-
formed autopsies and live dissections on criminals during the third and fourth century
BC2.
PMI determination not only establishes how long a person has been deceased but
also assists in distinguishing ante-mortem pathology from post-mortem artefact. The
accurate determination of PMI is essential for including and excluding suspects based
on their whereabouts at the time of death and helps to provide a timeframe in which
remains decomposed beyond recognition might be connected with missing persons.
Of the current results published for PMI determination, the majority can be divided
into two main categories: those related to the early post-mortem period and those
*Corresponding author. Email: andrea.donaldson@otago.ac.nz
2013 Australian Academy of Forensic Sciences

Australian Journal of Forensic Sciences 9
related to the late post-mortem period. Swift3 denes the early post-mortem period as
the soft tissue phase of decomposition; the late post-mortem period as skeletalisation
and alterations to the bony matrix3. Therefore, the early post-mortem period is dened
from death until the beginning of soft tissue decomposition or up until the bloat stages
of decomposition rather than any number of particular hours.
Many physico-chemical changes begin to take place in the body immediately after
death and progress sequentially until the body completely decomposes4. These physico-
chemical changes are typically described in ve stages of decomposition, fresh, bloat,
active decay, advanced decay and dry remains5. The timing of these changes is where
PMI determination becomes problematic as the nature, or presence, of each stage of
decomposition is strongly inuenced by endogenous and environmental factors such as
temperature, humidity, age of the deceased, use of medication, and disease1,2,4,68
despite all decomposing bodies going through any decomposition stages9.
It is also important to note that the PMI may be preceded by a signicant agonal
period, which can alter the accuracy of the PMI determination. A recent detailed
description of decomposition and the chemical changes that occur during this process is
available10.
Biochemical and metabolic proles from body uids taken after death reect the
agonal period, supravital reactions (reactions occurring from the moment of death until
cellular functions cease), leakage from cell degradation, diffusion and decomposition of
biochemical markers. This means that biochemical proles from different body uids
can provide insights into the changing metabolic environment of the host. Such proles
can provide useful information regarding cause of death and have the potential to
provide PMI estimations if suitable post-mortem biochemical markers are identied,
studied in detail and well documented. In addition, the use of biochemical markers for
the estimation of PMI means the elimination of examiner bias, something that
conventional methods such as algor mortis, rigor mortis, and putrefaction changes suffer
from. However, traditional biochemical methods have relatively low sensitivity and
require specialist skills, are labour intensive and so have not been widely adopted in the
forensic setting11.
Recent developments in biochemical techniques are more sensitive and specic than
traditional observational methods, such as presence of rigor mortis, algor mortis and
stages of putrefaction, so are able to facilitate high throughput approaches, unlike these
older methods. This means that researchers are starting to identify biomarkers using
Nuclear magnetic resonance (NMR) and mass spectrometry (MS) methods that may
provide more accurate PMI estimations.
This review focuses on PMI determined using biochemical markers in the blood-
stream that change in the early post-mortem interval as well as the current biochemical
methodologies and techniques that are being used to nd PMI biomarkers. Studies have
been carried out regarding PMI determination in body tissues other than blood, such as
cardiac tissue12,13, skeletal muscle14, pineal bodies15, pancreas16 and brain17 just to
name a few, but these studies will not be discussed in this review.
2. Research into estimating PMI using blood

The earliest recorded method for estimating the early PMI was a rate-of-change method-
ology based on the most easily observed morphologic changes. This was derived by the
Egyptians and Greeks who described and performed autopsies and live dissections on
criminals during the third and fourth century BC2. One of these methods, that of livor
10 A.E. Donaldson and I.L. Lamont
mortis, the reddish-purple hue on a corpse caused by settling of blood under gravity
within the capillaries and veins of a cadaver, is still used occasionally today, more than
2000 years later, but mainly to determine if a corpse has been moved rather than to
establish PMI. Photo-spectroscopy has been investigated as a method to quantitatively
measure the colour change of livor mortis in different tissues to determine PMI18,19.
However, this method proved inaccurate and unsuitable due to the large variations in
the occurrence, colour, and distribution of livor mortis in different corpses18,19.
Cadaveric blood collected and stored in blood tubes from corpses at 4C20,21 as well
as blood collected directly from corpses at room temperature22,23 to look at blood cell
morphology changes has been studied as a way to determine PMI. These studies dem-
onstrated that eosinophils, neutrophils, and monocytes displayed the earliest degradation
changes, typically within 46 hours post mortem. Lymphocytes were the most resistant
white cells to degradative changes and were identiable 120270 hours post mortem,
with very little degradation20,21 whereas the two other research groups reported that the
morphology of any cell beyond 3084 hour PMI was not identiable22,23. The reasons
for this difference were thought to be because of the temperature differences between
the experimental groups and because degenerative cellular changes are thought to occur
earlier and more rapidly in cadaveric blood compared with blood collected and stored
in blood tubes23.
Red blood cells and platelets have been examined post mortem by two research
groups. Both groups found that the morphology and shape of the red blood cells had
altered within 312 hours post mortem21,22. The second group reported that most red
blood cells had lysed by 20 hours post mortem22 where the rst group reported no lys-
ing of red blood cells by 168 hours even though the cells had altered shape by 12
hours21. The reason for this difference is most probably because of the methods that
were chosen to analyse the red blood cells. For example, the second group that reported
that most red cells had lysed by 20 hours22 examined the red cells from a blood smear
under a light microscope, whereas the rst group who found no lysis for 168 hours21
had buffered, dehydrated and covered the cells with egg white, carbon and gold before
looking at the cells under an electron microscope. Therefore, the rst group is most
likely to have altered the properties of the red blood cells by preserving them prior to
the analysis, and this is why the results appear so extremely different.
Platelets were identiable at all time points up to 10 days post mortem by the rst
research group even though they had formed aggregates21. The second research group
reported that no platelets were noted in their experiments beyond 21 hours22. Again,
the reason for this difference is related to the methods used to analyse the platelets.
In all studies that examined blood cell morphology, the conclusions reached were
that although blood cell morphology does change over time, variables such as the tem-
perature and the persons health prior to death affect the rate and morphological change,
suggesting that the used of blood cell morphology to determine PMI may only be used
in conjunction with other PMI parameters2022. Furthermore, it is clear that the analyti-
cal method used to examine blood cells appears to affect the rate and morphological
change, meaning that blood cell morphology is not a good parameter to use to deter-
mine PMI.
Biochemical changes are used as indicators of various types of disease, necrosis, or
infarction ante-mortem. These changes are also applicable in the eld of forensic sci-
ence and forensic medicine. The use of post-mortem biochemistry is now standard prac-
tice along with conventional, physical methods to determine the PMI24. The
biochemical methods can be standardised and statistically assessed, and can involve
multiple markers in one methodology. In addition, unlike the traditional changes, such
as algor mortis, rigor mortis, livor mortis, blood cell morphology changes and putrefac-
tion, which are strongly inuenced by a large set endogenous and environmental fac-
tors1 that are not clearly dened, biochemical changes such as protein degradation and
metabolite production, are inuenced by a set of variables that are more clearly dened,
making interpretation of the results easier.
Early nineteenth-century biochemical methods, such as the Brouardel method, which
examined the shift in ammability of putrefaction gases in the early post-mortem inter-
val, and the Westernhoffer-Rocha-Valverde method, which examined the formation of
crystals in the blood formed after the third day of putrefaction25 were examined as a
way to determine PMI, but the ndings of both methods had limited success. Today,
biochemical methods investigate pathophysiological changes occurring in the death pro-
cess that cannot usually be detected by morphological methods such as the assessment
of volatile organic compound formation4,10,2628, changes in the concentrations of pro-

teins or the activity of enzymes4,9,16,27,29,30 and changes in the production of metabo-
lites31,32. Most of these biochemical changes have been examined in body uids such
as blood, cerebrospinal uid (CSF), vitreous humour, pericardial uid, and synovial
joint uid2,3,7,24,26,27,3337.
Historically, analysis of post-mortem blood was the most extensively studied
because blood carries most substances present in a body33, although vitreous humour is
being currently examined to determine PMI. This is mainly due to the fact that vitreous
humour is well protected and isolated from the blood stream and microbes. Thus, the
autolytic changes proceed more slowly compared with blood. However, new techniques
based on biochemical changes are more specic and more sensitive to the biomarker(s)
of choice, meaning the biomarkers can be analysed from a poor quality sample. This
has enabled a research shift back to examining blood biomarkers.
Blood has been studied extensively resulting in a human plasma proteome database
(http://www.plasmaproteomedatabase.org) and a human metabolome database (http://
www.hmdb.ca/) being created. These databases are currently available to access infor-
mation on different blood proteins and blood metabolites as well as the methods for
analysing them3841.
3. Blood as a tissue from a forensic perspective

Blood is a uid that normally consists of 45% erythrocytes, 54% plasma and 1% leuko-
cytes and platelets42. The plasma component of blood contains proteins and metabolites,
including those that leak into the bloodstream after cellular injury from damaged cells.
A key advantage of blood from a forensic perspective is that it is a remarkably uniform
biouid, which is largely unaffected by confounding factors such as age, gender, diet,
diurnal cycles and stress, making it ideal to use for PMI determinations.
Furthermore, blood has been shown to ow after death, due to post-mortem contrac-
tion of the left ventricle and arteries, diffusion and rigor mortis development43, so that
cellular proteins and metabolites from degrading tissues detected in the blood may
potentially be able to be used as time of death markers.
Research involving the use of blood in PMI studies suggests that blood should be
collected only from the femoral vein, especially if it is to be used for measuring bio-
chemical markers. Femoral vein blood has very slight post-mortem changes that occur
slowly over time, unlike heart blood33,37, which can give huge changes very rapidly.
Femoral blood also gives values that best approximate those found in a living
individual33,37 meaning results can be compared to an ante-mortem reference range to

determine if values are abnormal, which is important since a post-mortem reference
range for biochemical markers does not exist.
4. Differences between ante-mortem and post-mortem blood

The most abundant and widely studied proteins and metabolites in human blood ante-
mortem are summarised in Table 1. It is to be noted that Table 1 lists only a small
fraction of the proteins and metabolites present in the blood, with 7518 blood proteins
identied and annotated on the human plasma proteome website and 4229 metabolites
identied and annotated on the human metabolome database to date.
Blood proteins and metabolites that are known to change signicantly after death are
described in Table 2. The blood pH decreases from 7.357.45 ante-mortem to 55.5
post-mortem33,44 and the blood haematocrit (HCT) increases from 4045 ante-mortem to
4778 post-mortem21. The blood haematocrit is the ratio of red blood cells in the total
blood volume, usually expressed as a percentage. The differences in haematocrit and the
increase in uidity are mainly the result of the falling pH of the blood post mortem. As
decomposition progresses, post-mortem blood becomes acidic due to the accumulation
of carbon dioxide, lactic acid and other chemicals formed as a result of tissue break-
down45. This decrease in pH causes brinolysin enzymes to be activated and released,
especially from capillaries and from serous surfaces such as the pleura4649, which pre-
vent blood clotting, resulting in an increase in uidity (non-coagulation) of the blood.
Altered uidity of the blood post mortem is a commonplace observation and is seen
more often in sudden or suffocation deaths48,50 because vasoactive materials such as
adrenaline and histamine are rapidly released in these deaths, this triggers a plasminogen
activator to be secreted, which prevents clotting4749.
The increase of the haematocrit is attributed to loss of plasma into surrounding tis-
sues, rather than an increase in the volume of red cells1. The loss of plasma into the
surrounding tissues is due to the lack of ATP needed for the functioning of the Na+/K+
pump in cell membranes. This causes sodium to accumulate inside cells, accompanied
by an osmotic increase in water from the extracellular uid such as plasma, which leads
to cell lysis45. Once cells have begun to lyse, there is widespread leakage of intracellu-
Table 1. Ante-mortem blood characteristics.
Proteins Metabolites/hormones
Albumin Glucose
Haptoglobin Lactic acid
Fibrinogen Insulin
Serum amyloid proteins (A2- and A) Atrial diuretic hormone
2- glycoprotein Growth hormone
Lipoproteins (LDL/HDL) Erythropoietin
Coagulation factors (ii, V, VIII, XII and XIIIb) Uric acid
Complement factors (B, C1R, C2, C3, C4A, C5, C6 H, I, etc) Creatinine
Transferrin Thyroxin
Haemoglobins ( and ) Adenosine Triphosphate
Keratins (1, 2 and 9) Glycerol
1- antitrypsin Niacinamide
Plasminogen Adrenaline
Immunoglobulins Pyruvic Acid
Proteins and metabolites in ante-mortem human blood are listed. Data taken from Refs. 30, 34, 90.
Table 2. Early postmortem blood proteins and metabolites.
Proteins Metabolites/hormones
Fibrinolysins () Glucose () 27.8 mmol.L
Haemoglobins ( and ) (=) Lactic acid () 50-60 mmol.L
Lipoproteins (LDL/HDL) (=) Hypoxanthine () 11-61.5mol.L
Acid and Alkaline phosphatases20 fold from Bilirubin and other bile pigments() 3.412
antemortem () mmol.L
Lactic dehydrogenase () Adrenaline () except in fatal trauma where
it is ().
Immunoglobulins (=) Uric acid () 327-368mol.L
Xanthine ()
Thyroxin ()
Ammonia () 0.587-1.76mmol.L
Proteins and metabolites in postmortem human blood which show concentration changes in the rst three days
are listed along with the concentrations. Some proteins and metabolites have symbols indicating whether con-
centration is increased (), decreased () or remains the same (=) postmortem as the literature did not specify
the concentration just stated whether it had changed. Data taken from Refs 21, 33, 36, 51, 52, 69, 87.
lar components into the extracellular and surrounding tissues spaces. Some of these cel-
lular components have been used as biochemical markers to determine PMI and will be
described in detail in the biomarkers section.
There are also signicant differences in the concentrations of some proteins and
metabolites post mortem, as shown in Table 2. Markers such as lactic acid, glucose and
acid phosphastase show very marked changes. Lactic acid increased from 0.5
2.5 mmol.L to 5060 mmol.L within the rst 24 hours after death51. Glucose increased
from 48 mmol.L to an average of 27.8 mmol.L in non-diabetic persons within the rst
24 hours after death52. Acid phosphatase increased to 20 times the normal ante-mortem
level by 48 hours after death53. The increase in lactic acid and glucose is a reection of
hypoxia and the consequent inhibition of the citric acid cycle, where the increase in
acid phosphatase is related to the lysis of lysosomes, thereby releasing the acid
phosphatases into the bloodstream.
5. Methods to study biochemical markers in blood

Owing to the immense diagnostic potential of blood there has been a strong interest in
searching for biochemical markers in serum that can identify disease and illness. The
Human Plasma Proteome Database was established in 2002 with the help of 55 differ-
ent laboratories around the world39. Protein biochemical markers in human plasma have
been mostly studied by using chemical fractionation and electrophoresis separation
steps39. To date, the majority of proteins discovered were found by using a combination
of two-dimensional (2D) gels, fractionation and MS methods. The Human Metabolome
database was set up in 2004 to identify all the metabolites in the human body41. The
majority identied to date were discovered using analytical methods such as NMR, gas
chromatography (GC) or liquid chromatography (LC) coupled to MS. These methods
are described below.
5.1. Two-dimensional (2D) gel electrophoresis

Two-dimensional gel electrophoresis is a method whereby charged molecules such as
proteins are separated based on charge according to their rates of migration in an
electric eld when on a gel support. The large molecules are also retarded in the gel
support enabling the molecules to be separated on size as well as charge. Therefore, 2D
gels separate proteins in a mixture by both size and charge. The application of 2D gel
electrophoresis was used to study plasma proteins in the 1970s, which contributed to
the current knowledge about range and size of these proteins54. The 2D map of the
human plasma proteome attained in the mid 1970s54 is recognisably the same as a 2D
map of plasma proteins that can be obtained in the present day. It is thought that the
reason for this reproducibility is due to the high solubility of the proteins involved and
the distinctive glycosylation of specic proteins, which can be easily recognised39.
Two-dimensional gel electrophoresis is still utilised today, although some drawbacks
remain. 2D gels are labour-intensive, require large amounts of sample, and have a lim-
ited dynamic range. The limited dynamic range is the greatest restriction with regards
to nding biomarkers in plasma because plasma proteins can differ in abundance39 by a
factor of 1010. An example of this limitation is searching for extremely low abundance
proteins (present at ng/ml to pg/ml), which comprise less than 1% of the total plasma
proteome55 amongst proteins such as serum albumin that are a high percentage of the
total protein present. To address this limitation, several types of fractionation methods
(chromatographic and immunodepletion) and mass spectroscopic methods are now used
in conjunction with 2D gels to identify proteins or biochemical markers55,56. For
example, comparisons of total ante-mortem and post-mortem proteins analysed with a
combination of 2D gel electrophoresis and Matrix Assisted Laser Desorption/Ionization
Time-of-Flight (MALDI-TOF) MS have demonstrated signicant changes in metabolic
enzyme concentrations57,58 that would not be identied by 2D gels alone. Assessing
post-mortem changes in protein amounts has the potential to provide a means to assess
time since death, in many instances long before visible cellular changes occur.
5.2. Fractionation methods

The most common fractionation methods are chromatography-based methods. Chroma-
tography is a technique for the separation of mixtures. The mixture is dissolved in a
uid, termed the mobile phase, which carries it through a structure (normally a column)
holding another material termed the stationary phase. The stationary phase is typically a
gel or bead support which is able to bind the molecule of choice or retard molecules in
direct proportion to their size, causing them to fractionate. The separation is based on
differential partitioning between the mobile and stationary phases. Liquid chromatogra-
phy (LC) is a separation technique in which the mobile phase is a liquid. Gas chroma-
tography (GC) is a separation technique in which the mobile phase is a gas. GC and
LC use chemicals, antibodies, cations and anions to separate or remove proteins and
metabolites of interest from samples to assist with the dynamic range problem. This
step is critical with regards to plasma proteins as described earlier, with 12 of the most
abundant proteins (albumin, IgG, brinogen, transferring, IgA, IgM, haptoglobin, apo
A-I, apo A-II, 1-antitrypsin, 1-acidglycoprotein, 2-macroglobin) comprising over
96% of total protein content in plasma and potentially masking other low abundant pro-
teins39. Albumin is the most abundant protein in plasma and is a carrier of many other
proteins and compounds, such as lipids and amino acids. The removal of albumin may
therefore have a profound impact on the identication of the proteins present56. To try
to minimise the loss of these low abundant proteins, technologies such as low abundant
protein enrichment kits have been developed.
5.3. Mass spectrometry

Mass spectrometry (MS) is an analytical technique that works by ionising chemical
compounds to generate charged peptides and measuring their mass-to-charge ratios,
which is different for each compound. MS, especially tandem mass spectrometry (MS/
MS), is able to serve as both a method of separation/fragmentation as well as character-
isation of separated components from a sample59. The advantage of this method is that
low-abundance biomarkers that may have been removed with fractionation methods are
still able to be separated and characterised by MS methods as no removal of substances
occurs. This means that identication of low abundant biomarkers is possible, as well
as being able to provide a rapid technique to analyse large numbers of biomarkers59. A
limitation of mass spectrometry is the need for large protein sequence databases to
which comparisons between your protein of interest and all the proteins that have the
same sequences can be made. Another limitation is the phenomenon of co-suppression.
Co-suppression is where the signal of target compounds, that have low ionisation ef-
ciencies, are lost to the MS interface59. To address these problems MS is normally cou-
pled to a fractionation method and MS spectral libraries such as the 2005 NIST/EPA/
NIH Mass Spectral Library (http://www.nist.gov/srd/nist1.htm) have been developed.
5.4. Nuclear magnetic resonance

Nuclear magnetic resonance (NMR) is a technique based on the principle that many
nuclei have spin, and placement in a magnetic eld will cause the nuclei to resonate
with specic frequencies. The frequency is measured and processed in order to yield an
NMR spectrum for the nucleus concerned, thereby identifying the compound. NMR is
used extensively to determine metabolite biomarkers, as it can identify and quantify a
wide range of organic compounds in micromolar ranges60. For example, NMR was able
to identify, glucogenic amino acids, cis-aconitate, acetone, 3-hydroxybutyric and hypo-
xanthine in plasma and urine despite being in micromolar ranges61. Additionally, NMR
has been used to study post-mortem biochemical changes in perchloric acid extracts of
rat skeletal muscle and provides a reasonable estimate for PMI determinations62. Unlike
MS, NMR is non-destructive, so samples can be used afterwards for further analysis.
The major limitation of NMR for metabolite proling is its low sensitivity, making it
inappropriate for analysis of the metabolites that are present in low abundance59.
5.5. Microarray applications

Protein microarray applications have also been used to screen and identify biomark-
ers56,63, such as antibody screening in clinical specimens64 and biomarker discovery for
prostate cancer65. Protein microarrays are a library of antibodies or protein molecules
arrayed on a glass microscope slide. The array is then probed with a protein solution.
This type of protein array is also known as forward phase protein array. In recent years,
reversed-phase protein microarrays have been developed in addition to forward phase
protein arrays66. Reverse-phase protein arrays involve a specimen such as serum being
printed on to a glass slide and then probed with antibodies against the proteins of
interest. Each spot printed on to the glass slide represents one sample containing multi-
ple analytes, and therefore many samples can be analyzed simultaneously under identi-
cal conditions. The specimen is printed onto the slide in serial dilutions that allow for
quantication. Janzi et al.63 used reverse-phase protein arrays to quantify IgA in serum
samples from thousands of patients who had been diagnosed with immunological dis-
eases caused by a deciency in IgA. This method is appealing because it allows for
screening of large numbers of samples and controls, in a single experiment. There is no
need to purify proteins as would be the case for normal microarray platforms. Limita-
tions of this method include non-specic binding, resulting in false positive identica-
tions56.
From all of the methods listed above, the only methods that have not been applied
to PMI determination to date are microarrays. Currently, PMI determinations are
preformed using GC/MS or LC/MS as the methods of choice4,10,27. For a detailed
account of all the studies in PMI determination that used LC/MS or GC/MS see the
review article10.
6. Biochemical markers of PMI

Some biochemical markers remain remarkably stable after death while others show
varying degrees of change. Biochemical changes have been attributed to three factors:
the agonal period of anoxia, the continuation of biochemical changes in the early post-
mortem period, and the distribution of easily diffusible substances between erythrocytes
and plasma as well as between interstitial uid, tissue cells, and the blood67. The bio-
chemical markers that have shown change over time after death in the bloodstream will
be discussed. These biochemical markers are separated into two classes: metabolites
and proteins.
6.1. Metabolites
6.1.1. Sodium and Chloride
Post-mortem serum concentrations of sodium and chloride decrease by, on average,
1 mmol/L per hour for the rst 352 hours post mortem33,36,52,68,69. Serum concentra-
tions of sodium and chloride also vary ante-mortem based on hydration status, kidney
function and illness33,36,52,68,69. These two markers are not reliable enough to be used
as a PMI indicator, but rather as a measure of electrolyte imbalance or kidney dis-
ease33,36. Sodium and chloride are now measured only from the vitreous humour, rather
than serum33,36.
6.1.2. Potassium
Serum potassium levels rapidly increase over the rst 12 hours after death51,52,6771.
However, potassium is released immediately from cells upon death7,33,69. This means
that establishing a serum ante-mortem concentration is difcult if not impossible, mak-
ing the use of serum potassium as a biochemical marker unsatisfactory. As with sodium
and chloride, potassium is measured in the vitreous humour. This is because it has been
repeatedly shown to rise rapidly in the rst 612 hours after death and then linearly
after 24 hours33,36,69,7275. A large number of confounding factors still exist such as
temperature, sampling techniques, differences between each eye as well as the age of
the individual, and the duration of the terminal episode75. The studies of several investi-
gators also indicate that the concentration of vitreous potassium rises much more rap-
idly post-mortem in an infant than in an adult76,77. Vitreous potassium values from
individuals dying from a chronic illness are more erratic than those dying of an acute
illness75. To date, no reliable method for post-mortem interval estimation based on mea-
surement of potassium ions has been developed.
Various statistical approaches have been applied in an attempt to improve the accu-
racy in estimation. It has been suggested that using potassium ion concentration as the
independent variable will improve the post-mortem estimation78. However, a review in
2006 found that in a random sample of 492 cases, only 153 were within the predicted
post-mortem interval with the remaining 339 having a systematic overestimation78.
6.1.3. Calcium
Jetter67 reported that the plasma calcium concentration was relatively stable in the early
post-mortem period from 1224 hours postmortem67. Hodgkinson and Hambleton71
found that plasma calcium concentration increased rapidly within an hour postmortem,
from 4 mmol/L to reach a peak of 9.4 mmol.L ve hours later before decreasing back to
ante-mortem levels by 11 hours postmortem71. Fekete and Brunsdon79 found a wide
range of serum calcium concentrations within two hours to 36 hours postmortem79.

All of these researchers used different methods to analyse serum calcium concentrations
and that may explain why the ndings are so variable, indicating that blood calcium
levels are not a reliable indicator of PMI. However, the most recent study looking at
serum calcium concentrations was 40 years ago. It would be worth analysing calcium
concentrations with more modern methods to establish what changes are occurring post
mortem and if these changes can be used to determine PMI.
6.1.4. Magnesium
Serum magnesium values were reported to not be substantially increased during the
early post-mortem period if cell integrity was maintained67 and when haemolysis
occurred, magnesium concentrations rose rapidly to 1015 mmol.L67. This nding was
not supported by other studies that found an early and progressive rise in concentrations
of magnesium in blood52,71. The reason for this difference is not particularly obvious.
Magnesium concentrations in post-mortem serum from different sampling sites (right
and left heart, subclavian vein, external iliac vein) from 360 autopsy cases were exam-
ined and no evidence was found that serum magnesium concentrations increase after
death80 in context to other studies. The authors did show that magnesium concentrations
were higher in peripheral blood compared with cardiac blood in victims of re fatali-
ties80. The authors explained this result by suggesting that because skeletal muscle has
a higher concentration of magnesium compared with cardiac muscle; peripheral blood
concentrations were higher as an inuence of post-mortem breakdown of skeletal mus-
cle. Additionally, magnesium plasma concentration was shown to be increased speci-
cally in salt water drowning victims and methamphetamine poisonings80. This indicates
that magnesium may be a good biomarker for cause of death rather than for PMI.
6.1.5. Phosphate
A post-mortem increase was observed in blood for both organic and inorganic phospho-
rus67. Inorganic phosphate was reported to be present at ante-mortem concentrations of
0.60.9 mmol.L/L and rises within one hour post mortem to reach concentrations of
6.6 mmol.L/L by 18 hours after death67. Similar trends by other groups have been
reported, although no concentrations were mentioned69. Therefore, this may be a poten-
tial marker for estimating PMI. However, more research needs to be conducted to estab-
lish reference ranges and an average increase that inorganic phosphate rises to over time.
Organic phosphorus is not normally present in the bloodstream ante-mortem. In one

study, it was present early in the post-mortem period67. If organic phosphate is not nor-
mally present in the bloodstream ante-mortem then this would be a suitable biomarker
of PMI. However, further research regarding this biomarker is required.
6.1.6. Lactic acid

Lactic acid is the simplest hydroxycarboxylic acid and exists as two stereoisomers, D
and L lactate. Normal serum lactate concentration is 0.52.2 mmol/L and is considered
almost entirely L-lactate, as D-lactate is present only in nanomolar concentrations81.
L-lactate is produced via anaerobic glycolysis in skeletal muscle, liver and red blood
cells. D-lactate is produced in small amounts in the methylgloxal pathway through
amino acid breakdown and dihydroxyacetone phosphate catabolism82. Circulating L-lac-
tate and D-lactate are taken up by facilitated transport, by a family of monocarboxylate

transport proteins (MCTs) that are differentially expressed in cells and tissues. These
MCT's are known as cell lactate shuttles and permit lactate to be oxidised to pyruvate
through the actions of lactate dehydrogenase (LDH) within actively respiring cells83.
Circulating serum lactate in human heart blood has been studied post mortem and
increased in the blood 20-fold one hour after death and rose 5070 times higher than
ante-mortem levels by 24 hours51. Lactate has also been measured post mortem in cere-
brospinal uid69, vitreous humour84 and in organs such as the brain17. It has been sug-
gested that lactate concentrations in the bloodstream post mortem may be affected by
lactate diffusion from muscular tissues and because of blood glycolysis in the vessels,
which would make lactic acid an unsatisfactory marker for PMI69. However, further
research is required to determine if this is the case.
6.1.7. Hypoxanthine
Hypoxanthine is an intermediate in the purine catabolism pathway. In the presence of
oxygen it is oxidised by xanthine oxidase to uric acid, the end product of purine metab-
olism in humans. During tissue hypoxia including death, elevated hypoxanthine concen-
trations are found in plasma, cerebrospinal uid and urine8587. There is evidence that
the hypoxanthine concentration increases linearly for the rst 24 hours after death in
both humans and animals88,89 so that it could be a potential biomarker for determining
the PMI. The concentration of hypoxanthine in the vitreous humour has been used reg-
ularly at the institute of forensic medicine in Oslo for six years to determine time of
death (http://www.todbase.org), however, due to signicant differences in the hypoxan-
thine concentration found in each eye33 this method is not adopted more widely. With
advances in technology available for analysing metabolites in the bloodstream, hypo-
xanthine may become an excellent biomarker for estimating PMI.
6.1.8. Urea, creatinine and uric acid

Numerous studies37,51,52,69,79 have established that post-mortem serum urea concentra-
tions closely approximate ante-mortem serum concentrations irrespective of the duration
of the post mortem interval or extent of decomposition. The only exception was
observed in a study of post-mortem blood and pericardial uid, which found a marked
elevation in urea as well as creatinine, and uric acid concentrations in the blood of
delayed traumatic deaths but not other deaths90. The urea, creatinine and uric acid con-
centrations in pericardial uid increased hours later than in the bloodstream, indicating
that these biomarkers may be suggestive of prolonged survival time rather than PMI90.
Other studies on serum urea, creatinine and uric acid concentrations post mortem have
not been reported.
6.1.9. Ammonia
Ammonia is formed in nearly all tissues and organs by normal amino acid catabolism.
Ante-mortem, ammonia is rapidly removed from circulation by the liver, where it is
converted to urea, leaving only traces (5.87-11.74 mol.L) in the bloodstream91. During
decomposition of a corpse, the ammonia produced from amino acids and tissue degra-
dation accumulates over time as it is not removed by the liver. In the only study relat-
ing PMI and ammonia concentrations, the concentrations of ammonia in plasma
increased after death with a rapid rise after 8 hours69. However, the methods used, the
highest ammonia concentration reported and the time frame of the experiment were not
reported. Further research into the relationship between ammonia and PMI is needed.
6.1.10. Catecholamines
Post-mortem serum catecholamine levels in relation to the PMI have been investi-
gated92,93 and adrenaline and noradrenaline concentrations were increased with post-
mortem time. The use of catecholamines to determine PMI is considered unreliable due
to the immediate release of adrenaline and noradrenaline from the adrenal glands into
the blood stream during stressful situations, such as during the process of the agony
period34. In addition, asphyxiation can cause catecholamine release34,92,93 and adrena-
line is used as a rst line drug by paramedics during a cardiac arrest, therefore serum
adrenaline levels may be falsely elevated. Therefore, catecholamines are not a good
maker for determining PMI.
6.1.11. Ethanol
Within a few hours of death gut bacteria penetrate the portal venous system and, after
about six hours, contaminate the systemic vessels94,95. Once in the blood, glucose and
lactate provide the substrates for microbial ethanol production94 providing the potential
for ethanol to be a marker of PMI. High environmental temperatures after death, septi-
caemia, and severe trauma with wound contamination all provide conditions for micro-
bial ethanol synthesis94,95. Distinguishing between alcohol ingested in life and
microbial production of ethanol after death is a problem if ethanol is to be used as a
marker of PMI; therefore, it is important that ethanol measurements in post-mortem
blood are corroborated by analyses of other body uids94. This is because body uids
such as urine and vitreous humour will only indicate alcohol ingestion, not microbial
production of ethanol because microbes do not quickly penetrate the bladder or the eye.
One study reported that blood putrefying in vitro had a rapid breakdown of glucose
over three days and from then onwards ethanol began to appear in the blood from
microbial fermentation, attaining its highest level between day 101296. However, to be
used as a marker of PMI other body uids need to be collected to conrm that the pro-
duction of ethanol is from microbes rather than alcohol ingestion. This means that using
serum ethanol as a PMI biomarker will not by itself yield highly accurate results.
6.2. Proteins
6.2.1. Total proteins
The total amount of protein in the blood has been considered as a marker for time of
death but was found to be variable and not signicantly different from ante-mortem lev-
els37,52,79. The concentration of total protein tended to increase time-dependently but
not signicantly, and remained within the range of ante-mortem values37,52 whereas
another study had results that were too variable to draw conclusions from79. These
results suggest the total protein concentration in serum is not a good biomarker for
PMI.
Two enzymes provide potential biomarkers for PMI determinations and they will be
described below.
6.2.2. Aspartate aminotransferase

Aspartate aminotransferase (AST) catalyse the reaction that converts aspartic acid to
glutamate. AST is found in the heart and skeletal muscle, as well as liver, kidney, eryth-
rocytes, and brain tissue97. AST is presently used as an indicator of liver damage due
to necropsy or carcinogenesis as it is released from the tissues into the extracellular
space98 and this will also occur post mortem. The amount of AST enzyme released
from any tissue depends on the severity of cellular damage. Erythrocytes contain high
concentrations of AST and haemolysis can elevate AST concentrations in the blood
stream. The post-mortem increase in the serum concentration of AST has been reported
to be roughly linear for the rst 60 hours post mortem, so that a crude PMI estimate
was possible53.
6.2.3. Lactate dehydrogenase

Lactate dehydrogenase (LDH) catalyses the terminal step in anaerobic glycolysis, pro-
ducing lactate from pyruvate and regenerating NAD+ from NADH. LDH is a tetrameric
enzyme containing two distinct subunits, conventionally those designated H for heart,
and M for muscle. These two subunits are combined in ve different conrmations
and numbered LDH1 through LDH597. Elevated levels of different isoenzyme indicate
particular disorders such as myocardial infarction, pernicious anaemia, late stage muscu-
lar dystrophy, liver disease, skeletal muscle damage, and some cancers98. LDH concen-
trations increase in blood post mortem52,53. LDH activity in serum increases rapidly
within a few hours post mortem followed by a slower increase for the next two to three
days, reaching its highest concentration around the fourth day post mortem45.
Red blood cells are the main source of serum LDH so that its post-mortem increase
must be ascribed to red blood cell autolysis69. LDH therefore provides a preliminary
biomarker for estimating PMI.
7. Conclusions and future directions

The need for objective, scientically-based research has been the current focus of foren-
sic science over the last few years and the area of PMI determination is no exception.
A considerable amount of work has been carried out by scientists to accurately deter-
mine time since death. Algor mortis, rigor mortis, livor mortis, and putrefaction
changes, which are strongly inuenced by endogenous and environmental factors, have
been a routine tool for the estimation of post-mortem interval for many years despite
being imprecise and inaccurate. The recent developments of biochemical methods have
the potential to completely change approaches to the estimation of post-mortem interval
as they are starting to identify biomarkers that may provide a fairly accurate PMI esti-
mation10,14,28,32,99,100.
Although relatively few biomarkers have been investigated as potential PMI mark-
ers, it is clear that several have high potential, such as volatile fatty acids, amino acids
and metabolites4,10,32. Much of the research on the use of bloodstream biochemical
markers for investigating PMI was completed decades ago with very few articles
describing current biochemical markers in blood to determine PMI. This provides a lot
of scope for PMI researchers to revisit some of these biochemical markers and to re-
examine them using some of the more recent methods and techniques, such as NMR
and mass spectrometry, which are more sensitive and specic than methods used in the
past. This will be particularly important for biomarkers such as hypoxanthine, phospho-
rus, ammonia, AST and LDH, which showed a lot of promise decades ago, but have
not been studied in detail by newer methods. Additionally, our understanding of the
death process and of proteins and metabolites has improved immensely over the last
5060 years, so that it is likely that biochemical markers previously considered unsuit-
able for analysis for PMI, such as calcium, could be usefully revisited. New technolo-
gies may also identify potential markers never before considered and these tools enable
lots of markers to be examined at once. In all likelihood, a single chemical marker will
not be accurate enough to determine post-mortem interval72 and it will likely be a com-
bination of lots of chemical markers. If the potential of these analytical methods to
determine post-mortem interval is to be accepted, there is a need for solid understanding
of these methods, including well-developed protocols derived through proper validation
and trials.
Older methods used to assess these biochemical markers in blood as indicators of
PMI have failed to gain general acceptance because, for the most part, they failed to
produce precise, reliable, and rapid results as required by the forensic community11. It
is likely that in the future, with the availability of both the human plasma proteome
database and the human metabolome database and in conjunction with the huge techni-
cal advances in biochemical methods and technologies, that new biomarkers of interest
will be examined and identied, which may help to solve the 2000-year-old struggle to
pinpoint time of death.
Acknowledgements
The authors wish to thank Dr Stephen Cordiner, (ESR, Porirua) and Dr Rachel Fleming (ESR, Mt
Albert) for their kind suggestions and useful comments on the draft manuscript.
AED was supported by a Te Tipu Putaiao PhD Scholarship from The Ministry of Science
and Innovation, Wellington, New Zealand.
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This article was downloaded by: [University of Winnipeg]

On: 11 September 2014, At: 11:15

Australian Journal of Forensic Sciences

Estimation of post-mortem interval

To link to this article: http://dx.doi.org/10.1080/00450618.2013.784356

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Estimation of post-mortem interval using biochemical markers

Determining the post-mortem interval (PMI) or time of death is a critical step in

most homicide or un-witnessed death investigations. However, the various methods

*Corresponding author. Email: andrea.donaldson@otago.ac.nz

2013 Australian Academy of Forensic Sciences

2. Research into estimating PMI using blood

of volatile organic compound formation4,10,2628, changes in the concentrations of pro-

3. Blood as a tissue from a forensic perspective

individual33,37 meaning results can be compared to an ante-mortem reference range to

4. Differences between ante-mortem and post-mortem blood

Table 1. Ante-mortem blood characteristics.

Table 2. Early postmortem blood proteins and metabolites.

5. Methods to study biochemical markers in blood

5.1. Two-dimensional (2D) gel electrophoresis

5.2. Fractionation methods

5.3. Mass spectrometry

5.4. Nuclear magnetic resonance

5.5. Microarray applications

6. Biochemical markers of PMI

range of serum calcium concentrations within two hours to 36 hours postmortem79.

Organic phosphorus is not normally present in the bloodstream ante-mortem. In one

6.1.6. Lactic acid

tate and D-lactate are taken up by facilitated transport, by a family of monocarboxylate

6.1.8. Urea, creatinine and uric acid

6.2.2. Aspartate aminotransferase

6.2.3. Lactate dehydrogenase

7. Conclusions and future directions

metabolic changes in porcine brain tissue using 1H-MR spectroscopypreliminary results.

by potassium in vitreous humor. Forensic Sci Int. 1989;40(3):231243.

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