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Putrefaction influence on the interpretation of toxicological results
1
Heesun Chung, Ph.D
Graduate School of Analytical Science and Technology
Chungnam National University, Daejeion, Korea
Chungnam National
University(CNU)
25,000 students
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members
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schools
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GRAST : Master and Ph.D students , all scholarship
GRAST has diverse fields of forensic chemistry, biology, physics,
chemistry, materials, chemical engineering, electrical
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Seoul
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Overview
Putrefaction processes
Putrefactive influence on drug concentration
Putrefactive influence on alcohol
Postmortem redistribution (PMR)
PMR of methamphetamine
Putrefaction
Dead
Digestion stops
Metabolism stops
Blood flow stops
Breathing stops
Decomposition starts
Putrefaction
Post mortem destruction of
soft tissues of the body by
the action of bacteria or
endogenous enzyme or both
Change in tissue
Change in color
Development of gases
Liquefaction
Following death
Pallor mortis
Algormortis
Rigor mortis
Livormortis
putrefaction
Putrefactive process
Variable on
Ambient conditions
State of the corpse
Degradation and /or synthesis of xenobiotics by
bacteria
Postmortem processes
Death and autolysis
Fermentative pathway
Enzyme activity
Bacterial invasion of
the body
Agonalinvasion
Post-mortem migration
Death and autolysis
O2 available in the body is depleted
Cells become anaerobic
Fermentative pathway of ATP
production
ADP and lactic acid being produced
Intracellular
environment rapidly becomes
acidic
Activate hydrolytic enzy
mes
Denature larger molecule
Lactate level reach max at 32-48hr, pH level falls max at 96 hrs
Enzymes lose their activity
Cytochrome P450 system
metabolizes numerous xenobioticsubstances such as drugs
lose over 90% of its activity in the first 48 h after death
Acid and alkaline phosphatases, esterase and β-glucuronidase
slowly loseβ-glucuronidase convert glucuronide metabolites back to the parent drug
Succinic dehydrogenase and cytochrome oxidase
heart, kidney and liver in 24-36 h
Bacterial invasion of the body
Human body is generally a sterile environment
except for
-the gastrointestinal tract,
-the lungs,
-the oral cavity
- the vagina
life
- Bacteria within the body to spread
- Bacteria from external sources of body: soil, insects or other animals
death
Mechanism of bacteria move
- Before death
- the last hours of life the resistance of the blood to infection –lower--pathogenic bacteria
AgonalInvasion
-After death
-Bacteria are liable to spread from the intestines --Endogenous bacteria multiply and migrate the body
Postmortem Migration
Post-mortem migration
Marbling: due to bacteria diffusing through the bloodstream and breaking down hemoglobin in erythrocytes to various other pigments
Within a week at 20C, in air, extensive bacterial invasion occur
externally ‘marbling” is visible
first externally visible sign in a decomposing body
green discoloration of the right lower abdomen
Bacteria in the lower end of the gastrointestinal tract
invade the surrounding tissues and blood stream
Bacteria move
death• Bacteria in gastrointestinal tract
first• Cross gastrointestinal wall
few• Enter blood and lymph vessels
hours• Transmigrate the body
List of bacteria identified in
human post-mortem samples
Alcaligenes faecalis Proteus vulgaris
Bacillus cereus Providencia species
Bacillus species Pseudomonas aeruginosa
Bacteroides fragilis Pseudomonas species
Clostridium perfringens Serratia marcescens
Escherichia coli Shigella flexneri
Klebsiella aerogenes Staphylococcus aureus
Klebsiella pneumoniae Staphylococcus epidermis
Lactobacillus species Streptococcus faecalis
Micrococcus species Streptococcus pneumoniae
Proteus mirabilis
Putrefaction on drug
concentration
Drug concentration in death is
similar in life?
No, it is not
Reliable
Detectable
Factors affecting drug
concentration reported after death
Circumstances of death
Time since death
Alteration of the body for example by embalming or putrefaction
Position of the body during transport
Site of sample collection
Enzyme activity
Preservation of sample
Security of sample
Red blood cell/plasma partition
Chemical stability of sample after collection
Postmortem metabolism/chemical instability of drug
Bacterial Degradation
Postmortem drug synthesis
Drug redistribution
Analytical issues related to tissue samples
Bacterial degradation on drug
concentration
Bacteria
Use particular drugs and/or metabolite as a substrates and create a pathway for
concentration change
Enteric bacteria metabolize drugs and produce ethanol
Metabolic reactions by bacteria
Reductive
Nitro, N-oxide, Oxime, Thiono, Sulfur-
containing Heterocyclic and
aminophenoic compounds
decomposed rapidly
Bacteria
Metabolize
Sulfur-containing antipsychotics
(Chlorpromazine)
Nitirobenzodiazepines
(Clonazepam)
Benzisoxazole derivative
(Resperidone)
Only metabolites of the parent drugs detected
Postmortem degradation by
bacteria (Benzodiazepines)
7-amino reduced form
Nitrobenzodiazepines
enteric bacteria
Nitrobenzodiazepines
- clonazepam,
- nitrazepam,
– flunitrazepam
• the liver, lungs, myocardium, kidneys, and skeletal muscles
7-amino metabolites
Robertson and Drummer
Reducing reaction notsuppressed by adding NaF
Postmortem degradation of
Cyanide
a significant decrease with time in lungs, brain, liver, and kidneys
Cyanide
During the putrefactive process by Bacteria
Analytical issues?
Decomposition Influence on
analytical aspect of toxicology
Produce lower molecular weight compounds such as indoleand phenethylamine
Interfere in many of the initial screening techniques (immunoassay)
Decomposition
Co-extracted putrefactive compound -Mask or alter the way a
drug detected
More selective method needed such as GC/MS and LC/MS
Analytical aspect of toxicology
β-phenylethylamine
The most typical amine produced during putrefaction
Putrefaction and analytical aspect
of toxicology
Bacterial activity that occurs during putrefaction
• affect the validity of quantitative results at the time of death
Degradation of the blood
• unsuitable as an analytical sample
• liver or other tissues need to be used
Enzyme activity on Drug
concentration
Enzyme
Continue to work after death
Cause breakdown of cellular materials after released during autolysis
Continuing metabolic activities of
enzyme
Cocaine
Hydrolysis
Benzolyecgonine
(major metabolite in antemortem)
Enzymatic conversion
Ecgonine methylester
(accumulation by cholinestrase in PM
blood)
Putrefactive influence on alcohol
Alcohol Synthesis after death
Ethanol and other alcohol
Enzyme , bacteria, yeast, fungi
Carbohydrate and proteins of the body
Putrefaction by fermentation
Ethanol synthesis
Ethanol
Bacteria and yeasts
The presence of glucidic substrates (glucose or ribose)
Glucose is the primary substrate
Ethanol synthesis in tissues
Tissue
the tissues with high glucose storage
capacity
the liver, skeletal muscles, lungs, and
myocardium
Brain
a lesser extent
Urine
poor medium
Production of ethanol postmortem
Rat with Deuterated
ethanol
Sacrifice
Putrefaction over 4 days at 30C in moist
chamber
Decreased
indicating degradation
Rat with Non-deuterated
ethanol
Sacrifice
Putrefaction over 4 days at 30C in moist
chamber
Increased
indicating endogenous
production by bacteria
How much alcohol produced after
death?
Ethyl alcohol can rise to quite high concentrations as a
result of bacterial metabolism
In decomposed bodies
10 mg/dL to 130 mg/dL
alcohol was not detected in the vitreous
humor
post-mortem result
as high as 1.5 mg/ml in organ tissues
Study by Zumwalt et al
Was ethanol recent ingested?
5-hydroxytryptophol (5-HTOL
5-hydroxyindole-3-acetic acid (5-HIAA)
The ratio of serotonin metabolites 5-hydroxytryptophol (5-HTOL,
pmol) versus 5-hydroxyindole-3-acetic acid (5-HIAA, nmol) in
urine
If ethanol was not recently ingested and is from post-mortem
production, the ratio of 5-HTOL/5-HIAA will be below 15
Was ethanol recent ingested?
How to determine whether ethanol was recently
ingested or not
The presence of Ethyl glucuronide, a metabolite of
ethanol
only present in cases where ante-mortem
ingestion was known
is sensitive and specific biomarkers for recent
alcohol ingestion
How to discriminate AM ethanol
from PM?
antemortem postmortem
N- Propanolproduced
not lower than 5% of a postmortem
ethanol concentration
Things to know in postmortem
ethanol synthesis
Mannitol administration just before death could favor
postmortem ethanol synthesis.
Volatile compounds such as methanol, n-propanol,
isopropanol, n-butanol, and sec-butanol may also be
produced in postmortem blood
Postmortem redistribution (PMR)
What is PMR?
Major modification of drug concentration after death
The rearrangement of drugs and chemical components between tissues, organs and fluids after death
Occur in the early postmortem period, prior to the putrefaction stage
Postmortem redistribution(PMR)
period of time
site of sampling
Drug concentrations
The change of concentrations of drugs in the body depends on the time of death and sampling sites
Site of sampling and period of time
• Blood drug concentrations are site-dependent
• Blood from peripheral sites appear to be less affected.
• Blood samples should be obtained from peripheral sites such as femoral vein less affected by PMR
Blood site
SubclavianVein
Heart Chambers
Femoral Vein
Period of time
autopsy
2hrs
Study of PMR in History
1975: Holt and Bernstead Digoxin
Jones and Pounder: imipramine, desipramine, diphenhydramine, codeine and
paracetamol
1987: Jones and Pounder: PMR was coined
1990: Pounder, toxicological nightmare
1990: Prouty and Anderson: 69 drugs
1992-1995: Druid and Holmgren: 83 drugs among 15,000 samples
2012: Schulz et al : 1,000 drugs
Repetto and Repetto: 103 drugs
Are there drugs to be prone to
PMR?
Drugs have a high volume of distribution(VD)
3–4 L/kg a relative threshold
Dependent on the protein binding tendency and lipid solubility of drugs
Basic drugs (high affinities to lung, heart muscle, liver and show wide distribution area)
Basic drugs?
stop
• Oxygen
• ATP
• NA/K pumping
damage
• Cell membranes
• organelles
inhibit
• Binding of protein with drugs
pH lower
• accumulation of lactic acid by anerobicglycolysis
Diffuse
• Basic drugs diffuse outside the cell easily
When it occurs?
Drugs that are present in relatively high concentration in the stomach, intestines or other organs diffuse into
• the surrounding tissues, blood and other bodily fluids such as CSF or vitreous humor
Drugs have higher concentrations in heart blood compared to peripheral sites
• Prone to redistribution
Redistribution of Cocaine
Heart muscle have high concentration of drug relative to the blood
Diffusion occur along this concentration gradient
Heart blood never used for quantitative analysis
Examples of Redistribution
Overdosing with intravenous administration
Lower peripheral concentration of a drug in blood
Incomplete distribution throughout the body prior to death
How to predict the PMR?
Ratio of central to peripheral blood concentrations
(C/P ratio)
Dalphe-Scottth
C/P ratio
• 1 or ˂1
•Low PMR
• ephedrine, hydrocodone, hydroxyzine, metoprolol, procyclidine, and trifluoperazine
C/P ratio
• ˃1 up to a maximal of 21
•Prone to PMR
• diphenhydramineindicating higher concentration in central sample
C/P ratios
• 113 drugs
• 320 cases
Newer approaches to predicting
drug redistribution
Alterations in blood/liver concentration ratios
A liver/peripheral blood ratio
A liver/peripheral blood ratio
• ˂5
• little prone to postmortem redistribution
• ˃20–30
• redistribution was likely to take place
When to measure the
concentration after death?
It is very important to measure as soon as possible.
• little changehaloperidol, quetiapine,
and risperidone
• increasedchlorpromazine
• decreased9OH-risperidone
Olaf Drummer
Concentration of 273 of antipsychotic drugs at femoral blood
samples
on arrival and when the autopsy (often days later)
PMR and toxicology
PMR results in increased uncertainty
during the interpretation of postmortem
toxicology results
PMR of methamphetamine
Postmortem study in Korea
Postmortem study
Postmortem study
Concentration of MA and AM in
postmortem specimens
Concentration and ratio in blood
MA concentrations (mg/L) AM concentrations (mg/L)
average average
central blood
(C)
0.42-204.10 19.68 0.001-8.70 0.73
peripheral
blood (P)
0.11-194.40 12.92 0.001-7.30 0.53
C/P ratio 0.79-6.59 1.96 0.20-6.67 1.86
Concentration and ratio in Bile juice
MA concentrations (mg/L) AM concentrations (mg/L)
average average
Bile juice(B) 0.55-149.40 30.49 0.001-4.20 1.04
peripheral
blood (P)
0.11-194.40 12.92 0.001-7.30 0.53
B/P ratio 0.77-10.50 4.76 0.58-12.67 5.49
Results of methamphetamine
The concentration of methamphetamine detected in cardiac
blood was about 2 times (average 1.96, median 1.72) higher
than that detected in peripheral blood,
the concentration detected in bile juice was about 4 times
(average 4.76, median 3.60) higher than that detected in
peripheral blood
Study by Barnhart et al
(J. Anal. Tox 1999)
20 methamphetamine fatal cases ( death and autopsy
between 12- 36 hrs)
Peripheral blood samples from the femoral vein
Methamphetamine and amphetamine measured by
GC/MS
Deaths of methamphetamine
Considerable variation in C/P ratios
the peripheral blood concentration was lower than central blood samples in all studies.
Central blood increases with time after death
Barnhart FE, Fogacci JR, Reed DW. Methamphetamine – a study of
postmortem redistribution. J Anal Toxicol. 1999;23:69–70.
20
Me
tha
mp
he
tma
ine
de
ths
Peripheral concentration: 0.3-4.1 ug/ml
Central blood :0.04-8.95 ug/ml
C/P ratio : 1.3 to 5.0
McIntyre IM et at (J. Anal. Tox 2013)
Three medical
examiner cases
Postmortem
redistribution occurs
for both
methamphetamine
and amphetamine
3 M
eth
am
ph
etm
ain
ed
ea
ths
MA: peripheral to antemortem blood ratios 1.51 (± 0.049; n = 3)
AM : peripheral blood to antemortem blood ratios 1.50 (n = 2).
postmortem blood concentrations
are ∼1.5 times greater than
antemortem concentrations
Things to consider
Conclusion
1.The autolysis stage can result in residual enzymatic
activity continuing to metabolise drugs and poisons
2.By-products of decomposition are putrefactive
compounds which can interfere with the analysis of
drugs
3.Peripheral blood samples should be analyzed for
quantitative purposes.
4.Care should be taken when interpreting quantitative
drug and poison results in forensic toxicology,
especially where there is evidence of putrefaction.
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