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10]

Review Article

Vitreous Humor: A Review of Biochemical Constituents in

Postmortem Interval Estimation
Weichen Li#, Yunfeng Chang#, Zijia Cheng#, Jiang Ling, Leiming Han, Xingmei Li, Yanjun Ding
Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha, China
#
These authors contributed equally to this work

Abstract
Postmortem changes in the biochemical constituents of the vitreous humor have been widely used to estimate the postmortem interval (PMI) over
the past several decades. However, few reviews have summarized the relationship between the postmortem vitreous biochemical constituents
and time of death. Herein, the relationship between PMI and single biochemical components, including vitreous potassium, hypoxanthine,
and amino acids, as well as comparisons of each statistical parameter in the formula, is summarized. We also discuss other compounds such
as urea and uric acid, which have no direct relationship with PMI. Utility of multiple constituent simultaneous analysis for estimating PMI
is being increasingly investigated. The promising idea of using simultaneous analysis of multiple constituents to determine PMI is proposed
as a future research direction.

Keywords: Biochemical constituents, multiple constituent analysis, postmortem changes, postmortem interval, vitreous humor

Introduction VH substances in forensic medicine mainly involve PMI

Postmortem interval (PMI) is the time that has elapsed after
a human has died.[1] It is used to predict the range of time of
death and narrow the scope of suspects in forensic analysis.
Accurate determination of PMI remains a challenge in
forensic medicine worldwide.[2] Although the exact PMI is
difficult to determine using a single postmortem method, an
approximate range of PMI can be estimated by combining
various physical and chemical methods.[3] Methods such
as evaluation of postmortem phenomena (algor mortis and
livor mortis), temperature‑based methods, microbial assay,
ocular changes, entomology, and others are applied for PMI
estimation.[4,5] Currently, using the postmortem changes in
body fluids (vitreous humor [VH] and blood) to predict PMI
has received attention in forensic medicine.
The VH is a colloidal substance between the lens and retina
which occupies 80% of the posterior chamber of the eye to
keep the retina in place.[6] VH is composed of gel‑like collagen
fibrils and fluid and is approximately 4–5  ml in volume.[7]
It contains 99% water and few solid substances composed
of macromolecular and low‑molecular‑weight constituents,
such as salts, sugars, and proteins.[1,8] The applications of
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estimation, postmortem identification, and cause of death
determination such as diabetes mellitus, sudden infant death,
hyponatremia, or hypernatremia.[6,8]
VH, because of its anatomically isolated site, is fairly stable
over a comparatively long period of time and is rarely
influenced by microbiological contamination, making it
very popular for analysis by forensic scientists.[9,10] It is well
established that the chemical composition of the VH is stable
and not greatly influenced by postmortem changes compared
to the blood and cerebrospinal fluid.[11,12] According to previous
studies, VH is an ideal postmortem specimen for investigating
postmortem changes in biochemicals, which are correlated
with PMI.[13] Therefore, the VH can be used for widespread
applications for PMI estimation in forensic medicine.
Address for correspondence: Dr. Yanjun Ding,
Department of Forensic Science, School of Basic Medical Sciences, Central
South University, Changsha 410013, Hunan, China.
E‑mail: dingyanjun@csu.edu.cn
This is an open access journal, and articles are distributed under the terms of the
Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which
allows others to remix, tweak, and build upon the work non‑commercially, as long
as appropriate credit is given and the new creations are licensed under the identical
terms.
For reprints contact: reprints@medknow.com

DOI: How to cite this article: Li W, Chang Y, Cheng Z, Ling J, Han L, Li X,

10.4103/jfsm.jfsm_13_18 et al. Vitreous humor: A review of biochemical constituents in postmortem
interval estimation. J Forensic Sci Med 2018;4:85-90.

© 2018 Journal of Forensic Science and Medicine | Published by Wolters Kluwer - Medknow 85
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Li, et al.: Vitreous humor: A review of biochemical constituents in postmortem interval estimation
Considerable progress has been made over the past several
decades in studies of VH for estimating PMI. Many
biochemicals in VH are used to estimate the PMI, and some
chemicals show a positive correlation. These compositions
can be divided into the following categories: electrolytes,[13,14]
amino acids,[15,16] hypoxanthine (Hx),[17] creatinine, urea
nitrogen, and uric acid.[7,18] Due to these previous investigations,
forensic examiners can accurately estimate PMI. Given this
background, the relationships between changes in the VH and
PMI should be further examined.
Electrolytes
Potassium
During life, intracellular potassium levels are maintained
at high concentrations, while extracellular (VH) potassium
levels are low because of the action of the Na/K‑pump.
After death, postmortem vitreous potassium levels change
because of cellular hypoxia, which induces the depletion of
Adenosine Triphosphate (ATP) and loss of selective membrane
permeability for ions, after which intracellular potassium
diffuses with the passive diffusion into the vitreous body,
leading to an increase in vitreous potassium levels.[19,20] This
mechanism, combined with cell autolysis, leads to changes
that cause postmortem levels to deviate from the antemortem
biochemistry of VH, which has been investigated in PMI
estimation studies.[21,22]
Since the first study revealed the linear relationship between
the PMI and potassium concentration in the VH, vitreous
potassium was considered as the most extensively studied
predictor for estimating PMI.[23,24] Over the past several
decades, many studies have confirmed the linear correlation
between vitreous potassium levels and PMI and calculated an
equation by linear regression analysis [Table 1]. For example,
Sturner,[20] a forerunner in VH analysis, reported 54 cases to
demonstrate the linear relationship between PMI and potassium
values. Madea et al.[25] reported a formula for PMI estimation
by determining vitreous potassium concentrations in 170
random samples, revealing a confidence interval of up to 120 h
postmortem. Thus, vitreous potassium may be used for a larger
range of PMI estimation in real cases compared to those used
in previous studies.
Table 1: Equation for estimating postmortem interval from vitreous potassium according to different authors
Author n Intercept (mmol/L)
Sturner[18] 54 5.6
Coe[2] 160 4.99
Madea et al.[25] 170 5.88
Munoz et al.[32] 133 5.35
Jashnani et al.[1] 120 2.616
Zilg et al.[26] 462 Unknown
Rognum et al.[27] 106 4.5
T: Ambient temperature, PMI: Postmortem interval
86 Journal of Forensic Science and Medicine  ¦  Volume 4  ¦  Issue 2  ¦  April‑June 2018
Recently, several new statistical methods have been applied for
PMI estimation.[12,26‑28] Many scientists demonstrated this linear
relationship between potassium and PMI with new approaches
and established different equations for estimating the time since
death with varying precision.[6] For instance, Rognum et al.[27]
conducted linear regression analysis to develop a formula that
takes ambient temperature into consideration. The lowest
standard error for potassium values was approximately 12 mM,
with the 95% limits of confidence interval approximately
at 3 h. Moreover, Lange et al.[28] reported precise results
(95% confidence limits of ± 1 h in the early PMI and 95%
confidence limits of ± 10 h, 110 h postmortem), which
have even exceeded optimistic expected results obtained in
previous investigations. These studies confirmed the utility
of potassium in VH as an index for estimating the time since
death. Ortmann et al.[14] studied five different equations for 600
random samples to improve the accuracy of PMI estimation
and obtained positive results by detecting vitreous potassium.
Although the researches described the above reported different
95% confidence limits of standard errors and equations for PMI
determination, the results showed agreement in the linearly
increased relationship between postmortem vitreous potassium
and PMI.[2,8,14,23,27,28]
In addition, numerous new analytical approaches have been
used to analyze the potassium level in the VH for PMI
estimation in recent years.[29-31] For example, Bortolotti et al.[30]
used capillary ion analysis to determine the vitreous potassium
levels in 164 cases to re‑evaluate the correlation with PMI for
2–110 h. Although some breakthroughs have been made in PMI
estimation during the first 24 h postmortem, the standard errors
of the values are less precise than those obtained using classical
methods. Our research team[31] recently explored a highly
sensitive fluorescence method based on silver nanocluster
probes for determining the VH potassium levels, which
showed improved potential for estimating the PMI in forensic
science. Compared to conventional detection approaches, our
new approach is easy, convenient, and sensitive for analyzing
vitreous potassium.[29,31-34]
Potassium levels may be influenced by various factors such
as the cause of death, season of death, and refrigeration of
the sample.[1] The impact of both objective and human factors
PMI Formula
104‑h postmortem PMI=7.14 (K7+) − 39.1
100‑h postmortem PMI=6.15 (K+) − 38.1
120‑h postmortem PMI=5.26 (K+) − 30.9
40‑h postmortem PMI=3.92 (K+) − 19.04
50‑h postmortem PMI=1.076 (K+) − 2.815
409‑h postmortem
PMI =
(
ln ( M − C 0 ) / ( M − [ K + ]) )
L 0 + mAA + mTT
118‑h postmortem PMI=5.164 (K+) + 0.174 T + (K+) × T × (−0.100) × 19.588
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Li, et al.: Vitreous humor: A review of biochemical constituents in postmortem interval estimation
should be considered, such as blood contamination from an
incautious puncture of blood vessels.[25,35] Among these factors,
some researchers reported that ambient temperature was the
most important factor in vitreous potassium determination.[3,27]
Thus, accurate equations for PMI estimation were obtained by
assessing various factors including ambient temperature and age.
Recently, multiple constituent analysis in VH has been
gradually developed to determine PMI.[1,28] For example,
some researchers found that the concentrations of vitreous
sodium decreased slowly after death, while potassium
gradually increased, and the relationship between the ratio
of sodium/potassium and PMI was explored.[1,10] In addition,
some studies used multiple compound analysis to estimate the
PMI, where the equation for PMI estimation was calculated
with relatively high precision.[28] To determine the exact PMI,
researchers utilize multiple compound analysis and integrate
all data to ensure precise estimation.[1,7,10,28] In the future,
the postmortem changes in VH can be evaluated by using
various compounds and influencing factors by simultaneously
monitoring multiple compounds using novel detection
techniques such as fluorescent‑sensing analytical techniques.[31]
Therefore, simultaneous analysis of multiple compounds may
become a main research focus in the future.
Sodium and chlorides
The sodium ion is the most abundant positive ion in the
extracellular fluid. The crystal osmotic pressure mainly
depends on the serum sodium concentration, and the volume of
the extracellular fluid compartment is determined by the total
body sodium content.[36] Chloride ion is the major anion in the
extracellular fluid and is closely related to the metabolism of
sodium and acid–base balance changes in the body.[37,38]
Postmortem changes in vitreous sodium concentrations
were used for postmortem diagnosis after at an early time of
death.[39] Several previous studies reported that the sodium
and chloride concentrations in VH marginally decreased
with increasing PMI in the early postmortem period.[10,40]
Other authors found that postmortem vitreous sodium
concentrations reflect abnormalities in the antemortem
concentrations of the serum sodium, enabling diagnosis of
hypo‑or hyper‑natremia.[41,42]
Subsequently, vitreous sodium and chloride have attracted
attention, with some researchers suggesting that sodium and
chloride levels in the VH can be used for PMI estimation.[43,44]
However, many studies found no relationship between PMI
and vitreous sodium and chlorides.[1,10] It is currently thought
that the concentrations of vitreous sodium and chloride have
no role in PMI estimation. Nevertheless, Zilg et al. showed that
both sodium and chloride levels in the VH slowly decreased
by approximately 2.2 mM/day as the PMI increased.[42] This
may be because of the small sample size of their study and the
decreasing concentrations of vitreous sodium change with a
narrow range of PMI. Therefore, compared to PMI estimation,
vitreous sodium and chloride values may be more useful for
determining the cause of death.[45,46]
Journal of Forensic Science and Medicine  ¦  Volume 4  ¦  Issue 2  ¦  April‑June 2018
Magnesium
Magnesium is an important marker for investigating postmortem
biochemical changes. Under antemortem conditions, a small
amount of magnesium diffuses from the retina to the lens and
into the VH. After death, ATP consumption and the loss of
selective cell membrane permeability lead to the redistribution
of magnesium ions between the extracellular fluid and
intracellular fluid in postmortem samples.[15] Therefore, the
mechanism of the increase in postmortem magnesium can be
used for PMI estimation.
It remains controversial whether vitreous magnesium can be
used as a predictor for PMI estimation. Previously, vitreous
magnesium levels were used to estimate PMI early after
death.[47] Farmer et al.[48] found a positive correlation between
PMI and magnesium levels over a limited range. However,
another study strongly disproved this conclusion regarding
the use of vitreous magnesium for estimating PMI.[15,49]
Currently, most forensic scientists consider that there is no
correlation between these two parameters. According to
many studies, it is possible that vitreous magnesium levels
can be used to estimate some specific causes rather than PMI
estimation.[15,49‑51]
Hypoxanthine
Hx is formed by hypoxic degradation of adenosine
monophosphate and may be elevated because of antemortem
hypoxia.[52,53] Postmortem Hx continues to increase in the
VH after the cessation of metabolism and its levels may be
correlated with an increasing postmortem time. Thus, Hx is
expected to replace vitreous potassium for determining the
PMI, as the changes in postmortem potassium are thought to
be easily affected by ambient temperature.[25,54]
In 1991, Rognum et  al.[18] first introduced Hx as a new
marker for estimating the PMI and found a strongly linear
correlation between PMI and Hx concentrations in 87 cases
without hypoxemia, where the Hx in the VH was detected by
high‑performance liquid chromatography (HPLC). Moreover,
the Hx formula for estimating the PMI was more precise
than that of potassium, particularly in the initial period
(the first 24 h) after death. They re‑examined this relationship
by combining the ambient temperature with K+ and Hx levels
in the VH. The observed PMIs showed an excellent correlation
with the regression models, and the lowest standard error for
Hx values was approximately 150 mM, corresponding to a
95% limit of confidence interval of approximately 2.5 h.[28]
In addition, other authors reported this correlation between
the vitreous Hx and time of death and obtained formulae, but
still observed the effect of ambient temperature on the Hx
increase in VH.[55,56]
According to previous studies, different factors such as the
cause of death, ambient temperature, and different analytical
methods may influence the Hx detection results and accuracy of
PMI estimation.[57,58] Among these factors, ambient temperature
remains the most important factor influencing Hx levels and
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Li, et al.: Vitreous humor: A review of biochemical constituents in postmortem interval estimation

formula slopes.[18] To further develop precise equations for
estimating the PMI, these intra‑ or extra‑factors should be
considered.[35,55]
Nitrogenous Compounds
Urea nitrogen, uric acid, and creatinine are the products of protein
metabolism and are considered as relatively stable compounds
in postmortem serum samples.[59,60] Many researchers have
examined nitrogenous compound levels in various biological
specimens (serum, pericardium, cerebrospinal fluids, synovial
fluid, and VH) collected during autopsy.[61,62] Several authors
investigated vitreous nitrogenous compounds to determine
postmortem changes in nitrogenous compounds and study
postmortem vitreous nitrogenous compound levels in relation
to various causes of death.[7,11,19]
In the early 1959, the concentrations of urea nitrogen and
creatinine were studied in the VH as an exploratory study to
evaluate their postmortem changes.[63] Subsequently, Coe[22]
compared the levels of vitreous urea nitrogen obtained from
each eye at different PMIs and found very stable values.
However, Hanna et al.[64] found a strong correlation between the
concentrations of urea and creatinine in serum and postmortem
VH in dogs over a 24‑h PMI, and the positive relationship in
previous studies may be related to limitations of the detection
method or some other factors. Finally, Palmiere et al.[19] found
that these parameters showed no PMI‑related differences in the
VH and other investigated fluids (e.g., postmortem pericardial
fluid and serum), confirming the biochemical stability of
nitrogenous compounds. In summary, most studies showed that
the vitreous creatinine, uric acid, and urea concentrations were
not correlated with the PMI.[24,19] These authors found that urea
and creatinine are very stable compounds in biological fluids
and that their postmortem values remained nearly unchanged
during storage.[7,19,40]
Amino Acids
Amino acids are the basic elements of large molecular proteins
and participate in a series of biochemical reaction processes
such as biosynthesis and catabolism. Some 300 additional
amino acids have already been discovered in cells and have a
variety of functions in humans. In their free form, amino acids
are transported across the blood–vitreous barrier.[65] This is the
basis of the maintenance of vitreous amino acid concentrations.
Therefore, numerous studies have focused on postmortem
changes in amino acids in VH and these molecules are considered
useful biomarkers for estimating the PMI [Table 2].[16,66]
In a previous study, Erdei and Vass[67] first reported the
existence of free amino acids in the VH. Subsequently, Patrick
and Logan[68] reported that the concentration of 27 amino acids
in VH had a linear relationship with PMI at different rates.
Girela et al.[16] determined vitreous free amino acid levels by
HPLC and the results showed that the levels of most amino
acids in the VH increased linearly as the PMI increased.
Niha and Shobhana[17,66] analyzed amino acids in the VH
using the new determination method. In the first study, they
reported the application of sensing technique‑based silver
nanoparticle fluorescence probe for determining the PMI by
quantifying vitreous cysteine (Cys). The equation determined
by using regression analysis was found to be PMI = 26.69
+  (−0.05) ×  (Cys).[66] In the second study, they utilized a
highly sensitive method based on fluorescence spectroscopy
to determine VH tryptophan levels. The results suggested
that tryptophan has a strong positive linear relationship with
PMI and revealed a gradual increase in VH tryptophan (Trp)
with increasing PMI up to 90 h, with a standard error of 5.2 h.
Moreover, the study reported that the regression equation was
PMI = 0.34 (Trp) − 0.74.[17] Using amino concentrations to
determine the time of death is becoming increasingly common
in forensic science. Although the utility of vitreous amino acids
is inferior to that of vitreous potassium, studies have revealed
the development potential of VH amino acid determination
for PMI estimation.
Conclusion
Much progress has been made in using the VH to estimate the
PMI [Table 3]. Vitreous potassium remains the best marker
for PMI determination. Many scientists have focused on
new statistical and analytical methods to accurately estimate
PMI. Utilizing Hx and amino acids for PMI determination is
becoming increasingly common in forensic science.
As a current research hot spot, multiple constituent simultaneous
analysis may be advantageous for immediately monitoring
postmortem changes of VH compounds. In the future, PMI
estimation studies with many biochemical constituents such as
ions, amino acids, glucose, and proteins may be simultaneously
analyzed by analyzing multiple constituents using novel
detection techniques such as fluorescent‑sensing analytical
techniques. As an analogy to the microarray technology,

Table 2: Relationships between postmortem interval determination and vitreous amino acid concentrations
Author n Selected amino acids Analytical method Findings
Erdei et al.[67] 1 13 (Cys, etc.) Chromatogram Identified the presence of free amino acids in VH
Patrick et al.[68] 120 27 (Taurine, etc.) Amino acid analyzer Linear relationship with PMI
Girela et al.[16] 58 19 (Trp, Leu, etc.) HPLC Linear relationship with PMI
Niha et al.[17] 90 1 (cystine) UV‑vis spectrophotometer PMI=26.69 + (−0.05) × (Cys)
Niha et al.[66] 76 1 (tryptophan) Fluorescence spectra PMI=0.34 × (Trp) −0.74
PMI: Postmortem interval, HPLC: High‑performance liquid chromatography, VH: Vitreous humor, UV: Ultraviolet

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Li, et al.: Vitreous humor: A review of biochemical constituents in postmortem interval estimation

Table 3: The relationship between biochemical constituents in vitreous humor and postmortem interval
Analyzed marker Routine analytical method Current conditions References
Potassium Flame photometry and ion PMI up to 120 h (especially ± 1 h in the early PMI) [1,18,20,21,25]
selective electrodes
Sodium Ion selective electrode No PMI‑related difference [8,38‑42]
Magnesium Ion selective electrode No PMI‑related difference [15,45‑47]
Hypoxanthine HPLC PMI=Hx × 0.215 + T × (−0.467) + Hx × T × (−0.005) + 10.353 [16,26]
Urea and uric acid Enzymatic uricase colorimetric Stable postmortem changes [5,17,20,58‑62]
Creatinine Enzymatic uricase colorimetric Stable postmortem changes [5,17,20,60‑62]
Amino acids Many new sensitive methods Have a linear relationship with PMI [16,63‑65]
T: Ambient temperature, HPLC: High‑performance liquid chromatography, PMI: Postmortem interval, Hx: Hypoxanthine

different fluorescent probes may be constructed for a matrix 2005;151:139‑49.

in simultaneously detecting most biochemical compounds in
the VH. To improve the accuracy of PMI estimation equations,
setting up a scoring system for multiple constituent simultaneous
analysis to evaluate the results of each single component and
consider various causes of death, states of illness, and influencing
factors can be helpful for estimating PMI.
Financial support and sponsorship
This research was supported by the National Natural Science
Foundation of China (No. 81772025) and the Natural Science
Foundation of Hunan Province (No. 2017JJ3511).
Conflicts of interest
There are no conflicts of interest.
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