Putrefaction influence on the interpretation of toxicological results

Heesun Chung, Ph.D Graduate School of Analytical Science and Technology Chungnam National University, Daejeion, Korea 1 Chungnam National University(CNU)

 25,000 students

 1,000 faculty members

 16 colleges and 13 graduate schools GRAST The No.1 National University Graduate School of Analytical Science and Technologyin Global Competitiveness

. GRAST : Master and Ph.D students , all scholarship . GRAST has diverse fields of forensic chemistry, biology, physics, chemistry, materials, chemical engineering, electrical engineering and etc. Where is? CNU in Daejeon

Seoul

City of Science Daejeon 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 Following death soft tissues of the body by the action of bacteria or Pallor endogenous enzyme or both mortis

Algor mortis  Change in tissue Rigor  Change in color mortis  Development of gases Livor  Liquefaction mortis

putrefaction Putrefactive process

 Variable on  Ambient conditions  State of the corpse

 Degradation and /or synthesis of xenobiotics by bacteria Postmortem processes

Bacterial Death and invasion of autolysis the body

Fermentative Agonal pathway invasion

Enzyme Post-mortem activity migration Death and autolysis

Fermentative O available in the Cells become 2 pathway of ATP body is depleted anaerobic production

Intracellular Activate hyd ADP and lac Denature larger environment ra rolytic enzy tic acid bein molecule g produced pidly becomes mes acidic Lactate level re ach max at 32-4 8hr, pH level fall s max at 96 hrs Enzymes lose their activity

Succinic dehydrogenase and cytochrome oxidase

heart, kidney and liver in 24-36 h

Acid and alkaline phosphatases, esterase and β-glucuronidase β-glucuronidase convert glucuronide slowly lose metabolites back to the parent drug

Cytochrome P450 system metabolizes numerous xenobiotic lose over 90% of its activity in the first substances such as drugs 48 h after death Bacterial invasion of the body

life death

Human body is generally a - Bacteria within the sterile environment body to spread except for -the gastrointestinal tract, - Bacteria from -the lungs, external sources of -the oral cavity body: soil, insects or other animals - the vagina Mechanism of bacteria move

Postmortem Agonal Migration Invasion

-After death - Before death -Bacteria are - the last hours of liable to spread life the resistance from the of the blood to intestines -- infection –lower-- Endogenous pathogenic bacteria multiply bacteria and migrate the body Post-mortem migration

Bacteria in the lower end of the gastrointestinal tract

invade the surrounding tissues and blood stream

first externally visible sign in a decomposing body

green discoloration of the right lower abdomen

Within a week at 20C, in air, extensive bacterial invasion occur

externally ‘marbling” is visible

. Marbling: due to bacteria diffusing through the bloodstream and breaking down hemoglobin in erythrocytes to various other pigments Bacteria move

• Bacteria in gastrointestinal tract death

• Cross gastrointestinal wall first

• Enter blood and lymph vessels few

• Transmigrate the body hours 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 . Red blood cell/plasma partition . Time since death . Chemical stability of sample . Alteration of the body for after collection example by embalming or putrefaction . Postmortem metabolism/chemical . Position of the body instability of drug during transport . Bacterial Degradation . Site of sample collection . Postmortem drug synthesis . Enzyme activity . Drug redistribution . Preservation of sample . Analytical issues related to . Security of sample 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)

Nitrobenzodiazepines Nitrobenzodiazepines - clonazepam, - nitrazepam, enteric bacteria – flunitrazepam

Reducing reaction not suppressed by • the liver, lungs, myocardium, adding NaF kidneys, and skeletal muscles

7-amino reduced form 7-amino metabolites

. Robertson and Drummer Postmortem degradation of Cyanide

Cyanide

During the putrefactive process by Bacteria

a significant decrease with time in lungs, brain, liver, and kidneys Analytical issues? Decomposition Influence on analytical aspect of toxicology

Decomposition

Produce lower molecular weight compounds such as indole and phenethylamine

Interfere in many of the initial screening techniques (immunoassay)

.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

The most typical amine produced during putrefaction

β-phenylethylamine 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

Enzymatic conversion Hydrolysis Ecgonine methylester Benzolyecgonine (accumulation by (major metabolite in cholinestrase in PM antemortem) blood) Putrefactive influence on alcohol Alcohol Synthesis after death

Putrefaction by fermentation

Carbohydrate and proteins of the body

Enzyme , bacteria, yeast, fungi

Ethanol and other alcohol Ethanol synthesis

The presence of glucidic substrates (glucose or ribose) Glucose is the primary substrate

Bacteria and yeasts

Ethanol Ethanol synthesis in tissues

the tissues with high glucose storage capacity the liver, skeletal muscles, lungs, and myocardium

Tissue

Urine Brain poor a lesser medium extent Production of ethanol postmortem

Rat with Rat with Non- Deuterated deuterated ethanol ethanol

Sacrifice Sacrifice Putrefaction over 4 Putrefaction over 4 days at 30C in moist days at 30C in moist chamber chamber

Increased Decreased indicating indicating endogenous degradation 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 post-mortem result bodies

10 mg/dL to 130 mg/dL as high as 1.5 mg/ml in organ tissues alcohol was not detected in the vitreous humor

 Study by Zumwalt et al Was ethanol recent ingested?

 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

5-hydroxytryptophol (5-HTOL

5-hydroxyindole-3- acetic acid (5-HIAA) 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- Propanol produced

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)

 The change of concentrations of drugs in the body depends on the time of death and sampling sites

period of time

Drug concentrations

site of sampling Site of sampling and period of time

Blood site Period of time

Subclavian autopsy Vein

Heart 2hrs Chambers

Femoral Vein

• 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 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

Repetto and Repetto: 103 drugs

2012: Schulz et al : 1,000 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 damage inhibit

• Oxygen • Cell • Binding of • ATP membranes protein with • NA/K • organelles drugs pumping

pH lower Diffuse

• accumulation of • Basic drugs lactic acid by diffuse outside anerobic the cell easily glycolysis 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 • 1 or ˂1 •Low PMR • ephedrine, C/P ratio hydrocodone, hydroxyzine, metoprolol, procyclidine, and • 113 drugs trifluoperazine C/P ratios • 320 • ˃1 up to a maximal of cases 21 •Prone to PMR C/P ratio • diphenhydramine indicating higher concentration in central sample Newer approaches to predicting drug redistribution

. Alterations in blood/liver concentration ratios

• ˂5 A liver/peripheral • little prone to blood ratio postmortem redistribution • ˃20–30 A liver/peripheral • redistribution blood ratio was likely to take place When to measure the concentration after death?

 It is very important to measure as soon as possible.

 Olaf Drummer

 Concentration of 273 of antipsychotic drugs at femoral blood samples

 on arrival and when the autopsy (often days later)

haloperidol, quetiapine, and risperidone • little change

chlorpromazine • increased

9OH-risperidone • decreased 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 0.42-204.10 19.68 0.001-8.70 0.73 (C) peripheral 0.11-194.40 12.92 0.001-7.30 0.53 blood (P)

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 0.11-194.40 12.92 0.001-7.30 0.53 blood (P)

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

deths Peripheral concentration: 0.3-4.1 ug/ml ratios . the peripheral blood Central blood :0.04-8.95 ug/ml concentration was lower than central blood samples in C/P ratio : 1.3 to 5.0 all studies. Methamphetmaine . Central blood 20 20 increases with time after death

Barnhart FE, Fogacci JR, Reed DW. Methamphetamine – a study of postmortem redistribution. J Anal Toxicol. 1999;23:69–70. McIntyre IM et at (J. Anal. Tox 2013)

MA: peripheral to antemortem blood ratios 1.51 (± 0.049; n = 3)  Three medical

examiner cases deaths AM : peripheral blood to antemortem blood ratios 1.50 (n = 2).  Postmortem redistribution occurs for both methamphetamine

postmortem blood concentrations and amphetamine Methamphetmaine

3 3 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.