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 Table of Contents  
Year : 2018  |  Volume : 4  |  Issue : 2  |  Page : 85-90

Vitreous humor: A review of biochemical constituents in postmortem interval estimation

Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha, China

Date of Web Publication29-Jun-2018

Correspondence Address:
Dr. Yanjun Ding
Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha 410013, Hunan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jfsm.jfsm_13_18

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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

How to cite this article:
Li W, Chang Y, Cheng Z, Ling J, Han L, Li X, Ding Y. Vitreous humor: A review of biochemical constituents in postmortem interval estimation. J Forensic Sci Med 2018;4:85-90

How to cite this URL:
Li W, Chang Y, Cheng Z, Ling J, Han L, Li X, Ding Y. Vitreous humor: A review of biochemical constituents in postmortem interval estimation. J Forensic Sci Med [serial online] 2018 [cited 2022 Nov 29];4:85-90. Available from: https://www.jfsmonline.com/text.asp?2018/4/2/85/235441

Weichen Li, Yunfeng Chang, Zijia Cheng. These authors contributed equally to this work

  Introduction Top

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 VH substances in forensic medicine mainly involve PMI 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.

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 Top


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

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Recently, several new statistical methods have been applied for PMI estimation.[12],[26],[27],[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],[30],[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],[32],[33],[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 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]


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],[50],[51]

  Hypoxanthine Top

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 formula slopes.[18] To further develop precise equations for estimating the PMI, these intra- or extra-factors should be considered.[35],[55]

  Nitrogenous Compounds Top

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 Top

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]
Table 2: Relationships between postmortem interval determination and vitreous amino acid concentrations

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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 Top

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.
Table 3: The relationship between biochemical constituents in vitreous humor and postmortem interval

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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, different fluorescent probes may be constructed for a matrix 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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  [Table 1], [Table 2], [Table 3]

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