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)

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members

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schools

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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.