biochemical changes of vitreous humor and its medicolegal importance

1. Biochemical changes in vitreous humour
By
Dr.Nafeeya
Department of Forensic Medicine and
Toxicology

2. Overview
Introduction
anatomy and physiology
Biochemistry of vitreous
Collection of vitreous in autopsy
Medicolegal autopsy

3. Introduction
• Postmortem analyses of different body fluids can help to determine the
cause and manner of death, as well as time of death.
• Blood can be used for toxicological analyses other body fluids that can be
used for postmortem are vitreous, cerebrospinal, pericardial and synovial
fluids.
• It is less affected by postmortem contamination and putrefaction.
• Vitreous fluid can be used to analyse electrolytes (sodium, potassium,
chloride) and a variety of endogenous compounds like acetone, urea,
hypoxanthine, glucose and lactate

4. anatomy and physiology
• The vitreous body is a colourless, transparent
gel that fills up the eyeball.
• It is also the hardest structure of the eye ,
transparent in order to let light travel freely
through the eyeball and hit the retina.
• It consists mainly of water (99%), collagen
and hyaluronic acid.
• No blood vessels in the vitreous
• Round structure of the eye,
• Shock absorber,
• To move solvents and solutes within the eye

5. Biochemistry of vitreous
• Vitreous is around 99% water , connective tissue and an extracellular matrix.
• The rest of the vitreous consist of salts (0.9%) and proteins and polysaccharides
(0.1%).
• Collagen
• Hyaluronic acid
• Cells
• Low molecular weight substances
• Bicarbonate, Proteins, Lipids And Amino Acids.

6. Collagen
• Collagen is the major structural protein of the
vitreous.
• Collagen is composed of three individual
polypeptides known as alpha chains, organised
in a triple helix configuration.
• Triple helixes join together and form collagen
fibrils, which in turn form collagen fibres
• Most of the collagen in the vitreous is type II.
• The vitreous base has the highest density of
collagen fibrils.

7. Hyaluronic acid
• Hyaluronic acid - glycosaminoglycan
(GAG).
• Unbranched molecule -twisted network.
Cells
• Cells normally found in the vitreous are
hyalocytes and fibrocytes.
• Hyalocytes are the most abundant (90%)
and are found in the vitreous cortex.
• They produce HA and function as
phagocytes
• Fibrocytes are primarily located in the
vitreous base and are believed to produce
collagen.

8. NATIONAL INSTITUTE OF HEALTH/NIH SCORING OF VITREOUS CELLS
Lowmolecularweightsubstances
Sodium – level are lower in vitreous fluid than in blood.
Potassium- levels rises in the vitreous after death.
Chloride- levels are slightly higher in vitreous fluid than in blood.
Calcium -levels are lower in vitreous fluid than in blood.
Glucose- levels are lower in vitreous fluid than in blood

9. collection of vitreous in autopsy
• Vitreous fluid is ideal .
• After death, blood cells in the eye break down
and release potassium. Slow process
• It also resists putrefaction longer than other body
fluids.
• Vitreous humor was collected at autopsy from the
posterior chamber of the eye.
• Liquid paraffin gel was injected in the posterior
chamber of eye for cosmetic purpose.
• -20°C for a maximum of 30 days post-mortem,

10. • The vitreous samples were analysed with
a blood gas instrument, ABL 625
(radiometer) by direct injection of the
content of the syringe.
• The lactate -Amperometric electrode
equipped with an enzymatically active
membrane
• Glucose and lactate with Standard
clinical chemistry technique based on
enzymatic conversion
• Different sampling techniques,

11. MEDICOLEGAL IMPORTANCE

12.  Estimation of time since death from vitreous glucose, lactate,
potassium and hypoxanthine levels.
 Interpretation of postmortem vitreous concentrations of sodium and
chloride to rule out cause of death.
 Estimation of vitreous alcohol concentration.
 Estimation of drug detection in vitreous .
 Metals and (metallo)proteins identification in vitreous humor focusing
on post-mortem biochemistry .

13. Age related changes in vitreous
• The vitreous of an infant is a homogeneous, gel-like substance.
• With age, the gel volume decreases and the liquid volume increases, a process that
is called vitreous liquefaction.
• At the age of 40 years, the vitreous is 80% gel and 20% liquid, and by 80 years it is
50% liquid.
• Both HA and collagen cause dissolution of the HA-collagen complex: collagen
fibrils move out of the collagen network, causing them to unite
• Fibres and then into bands.
• The redistribution of collagen leaves spaces next to these bundles, allowing
pockets of liquid vitreous called lacunae.

14. Influence of temperature in vitreous

15. • Postmortemanalysesof vitreous fluid
1. Glucose :
• The postmortem diagnosis of hyperglycemia.
• Vitreous glucose decreases immediately after death.
• Hypoglycemia is extremely difficult to diagnose by postmortem glucose
levels.
• Vitreous glucose levels decrease the first 12-24 hours after death and are
relatively stable after that.
• A vitreous glucose level of 10-26 mmol/L indicates severe antemorterm
hyperglycemia.
• Ketoacidosis-5000umol/L in vitreous

16. • LACTATE:
• Lactate concentrations increase 20-fold by one hour after death and 50-70-
fold by 24 h
• Loss of selective membrane permeability.
• Estimating The Time Of Death
• Lactic acid increases after death.
• Normal -80-160mg%,20 hrs-210mg %
• Increases with a long agonal period and also in cases of hypoxia caused by
shock (multi organ failure)
• For these reasons, the determination of the postmortem interval cannot be
obtained using only lactate analysis, further investigations are needed.

17. 2.Potassium
• The postmortem increase in vitreous potassium levels,
which can be used to estimate the time of death.
• There is linear relationship between vitreous potassium
concentration and time since death.
• Potassium levels were found to increase up to 120 hrs.
• Potassium values in burn cases are found to be higher
than the non-burn cases.
• The levels of potassium in vitreous humour are found
to be the same, when the vitreous samples from both
eyes were drawn at the same time and were examined
as separate samples.
• PMI=5.26 x k conc -30.9

18. Time since death 1st eye 2nd eye
Within 12 hrs 05.66 ± 1.76 06.78 ± 2.11
12.1 – 24 hrs 08.59 ± 1.06 09.42 ± 1.14
Above 24 hrs 12.88 ± 2.49 12.14 ± 1.98
Mean potassium concentration (meq/l) in relation to time since death
Non burns cases
Time since death 1st eye 2nd eye
Within 12 hrs 6.05 ± 1.59 07.20 ± 0.64
12.1 – 24 hrs 9.84 ± 1.51 11.24 ± 1.34
Above 24 hrs 13.09+1.60 15.00 ± 0.00
Burns cases

19. PMI IN HOURS POTASSIUM
LEVEL(meq/L)
0-6 5.1
6-12 5.5
12-18 6.4
18-30 6.9
30-42 9.2
>42 9.4
Potassium levels in time since death

20. • Hypoxanthine:
• In cases of sudden death caused by trauma or by myocardial infarction,
levels ranging from 7.6 umol/L
• The mean value was about ten times higher than normal plasma level
• The hypoxanthine levels seem to be independent of time post-mortem, at
least during the first 48 hours
• Hypoxanthine levels in vitreous humor: main factors in sudden infant
death syndrome
• Elevated hypoxanthine levels in the vitreous humor of the sudden
infant death syndrome (SIDS) have been claimed to indicate hypoxia
before death.

21. 3.Sodium and chloride
• Vitreous sodium and chloride levels slowly decrease with time of death
approximately 2.2 mmol/L per day after death.
• Postmortem vitreous sodium and chloride levels correlate well with
antemorterm serum sodium and chloride levels.
• Used to diagnose various antemorterm sodium/chloride imbalances, such as
dehydration or water intoxication.
• Sodium imbalances in cases of drowning are most likely due to postmortem
diffusion between water and vitreous, rather than the drowning process
• Insignificant

22. Age group Sodium Potassium Chloride Time since
death-hours
<20 138 8.9 121 11.9
20-29 137 9.3 122 12.00
30-39 131 9.1 118 12.30
40-49 129 8.6 116 11.70
50-59 139 9.4 121 13.50
>60 135 8.9 120 14.60
Vitreous electrolytes and time since death in different age groups

23. Estimation of vitreous alcohol concentration
• Gas chromotography method
• A Vitreous Alcohol Concentration [VAC]in excess of the Blood
Alcohol Concentration [BAC]
• Valid for only 36 hours-BAC
• Suggests that most deaths occurred during the elimination phase
• The BAC may significantly exceed the VAC if death occurs before equilibrium
is achieved (rapid death)
• Alcohol distributed in the tissue depends on water content.

24. Metals identification in vitreous humor
• Three metal ions (fe, mg and mo), were identify and quantified in the
vitreous humor samples with different PMI for decomposing bodies and
non-decomposing bodies
• The good correlation between fe and PMI, was clearly demonstrated due
to the presence of serotransferrin in vitreous
• Serotransferrin, indicating that its transport to the eyeball is probably due to
the disruption of the blood ocular barrier

25. • Drug detection
• Amphetamines, Cocaine, Opioids
• Enzyme immunoassay method
• The degradation and redistribution of drugs after death has caused several
challenges.
• Lower concentrations in the vitreous humor than in the blood.
• The vitreous humour matrix was found to be a viable alternative to blood and urine
in advance putrefaction .
• Disadvantages -limited specimen volume and the limited interpretative value of
analytical results.

26. REFERRENCE
• JIAFM 2004-26(4)-changes in the levels of vitreous potassium with
increasing time since death
• Forensic science international journal , 12 (1978) 33 - 36 Post-mortem
hypoxanthine levels in the vitreous humour.
• Journal Name-Metallomics Metals and (metallo)proteins identification in
vitreous humor focusing on post-mortem biochemistry
• Forensic Science International.Journal interpretation of postmortem
vitreous concentrations of sodium and chloride
• Journal of Forensic and Legal Medicine .An evaluation of sodium,
potassium and chloride levels in estimation of time since death (during the
first 36 h after death)
• Journal of analytical toxicology, vcl. 29, july/august 2005.Comparative
alcohol concentrations in blood and vitreous fluid
• Journal of analytical toxicology vol 32 oct 2008 -Rapid Detection of
Opioids in Vitreous Humor by Enzyme Immunoassay
• A.k. khurana & Indu khurana