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Thursday, October 9, 2014

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“Chemistry of Death”




Dr. Darren Griffin Professor of Genetics,

University of Kent

Lucas Zarwell Chief Toxicologist,

DC Medical Examiner's Office

“The Chemistry of Death” A Review of Postmortem Redistribution

in Forensic Toxicology

Lucas Zarwell, MFS, D-ABFT-FT

Chief Toxicologist

Office of the Chief Medical Examiner

Washington, DC

10/1/2014

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Postmortem Redistribution

Drug movements within the body after

death which cause time-dependent

variations in blood and tissue drug

concentrations prior to autopsy

Who Cares?

• Medical Examiners may depend on toxicology

results to help determine the cause and

manner of death

• PMR (Postmortem Redistribution) may be

misleading, attributing high drug concentrations

with a toxic effect

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Our Goal

To understand postmortem redistribution

in terms of chemistry, pharmacology,

and forensic interpretation

Please Ask Questions

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Case Example 55 year old male is found deceased in bed in a

secure residence. There is an antidepressant

medication on scene next to the bed including

the tricyclic antidepressant imipramine. In

addition there is 1/2 full bottle of wine on the

floor. At autopsy, the medical examiner can find

no immediate anatomical cause of death. The

medical examiner submits venous blood, heart

blood, vitreous humor and liver to the forensic

toxicologist for analysis.

Imipramine

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Ethanol

Let's Get Started

• Drug Chemistry ->

• Drug Pharmacokinetics ->

• Distribution Mechanisms ->

Discuss the major contributing elements to PMR

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Drug Chemistry

• Acid / Base Properties

(pKa)

• Lipophilicity

• Size and Structure

pKa • What is pKa?

• It is derived from Ka which is the equilibrium

constant for the chemical reaction known as

dissociation in the context of acid-base

reactions

• pKa = - log10

Ka

• Ka is a quantitative measure of the strength of

an acid in solution

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pKa • pKa is used in practice to avoid

the many orders of magnitude

spanned by Ka

• The value can be assigned to

both acids and bases

• Essentially: the smaller the pKa

the stronger the acid, the higher

the pKa, the stronger the base

• Thus one can determine the

degree of ionization at a given

pH

Strong Base

Strong Acid

What?

• When we substitute these elements (pH and pKa) in

to the Henderson-Hasselbalch equation we can

mathematically determine how much of a drug is

ionized at a biological pH

• The ionization of drug molecules is important with

regard to their adsorption into the circulation and

their distribution to different tissues.

• It's also handy when you are trying to extract on the

bench!

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Ok, What about our case?

The pKa of Imipramine: 9.5

The pKa of Ethanol is 15.9

The approximate antemortem pH of the small

intestine is 6

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Lipophilictiy

The nonionized form of

the drug tends to be

more lipid soluble.

Normal biological pH is

about 7.4

Imipramine is highly

lipid soluble

Lipophilicity

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Size and Structure

Imipramine:

280 Da

Formula: C19H24N2

Size and Structure

Calciseptine

(mamba venom):

7000 Da

Formula:

C299H476N90O87S10

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Drug Pharmacokinetics

• Drug Chemistry

• Protein Binding

• Volume of Distribution

• Storage Depots

Drug Chemistry

Passive Transport

Filtration (think Kidney)

Active Transport

Facilitated Diffusion

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Protein Binding Drug Chemistry effects

drugs ability to bind

with plasma proteins

and tissues in the

blood

• Albumin attracts

acidic drugs

• α1-acid

glycoprotein

attracts basic drugs

Volume of Distribution

Drug chemistry effects the

Volume of Distribution (Vd)

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Volume of Distribution

Vd is determined experimentally

𝑽𝒅 =𝑨𝒑

𝑪𝒑

Imipramine has a Vd of 20-40 L/kg

Ethanol has a Vd of <1 L/kg

Storage Depots

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High PMR

Low PMR

Vd

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These are elements of biochemistry which

influence drug blood and tissue concentrations

whether an individual is alive or dead....

Dead

• Digestion stops

• Metabolism stops

• Blood flow stops

• Breathing stops

• Decomposition starts

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Cell Death • Aerobic respiration stops

• Oxygen is no longer provided (hypoxia)

• In the mitochondria, oxygen was the final

electron receptor of the electron transport

system responsible for the synthesis of ATP

from NADH

• Thus we no longer have ATP to run cellular

operations - and cellular death begins

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Cell Death

Cell Death • Build up of lactic acid result in decreases in intercellular

pH

• Na begins to build up in the cell (ATPase pump has failed)

• Water is osmotically pulled into the cell AND increasing catabolites add the intracellular osmotic load

• Leads to cellular dilation, disruption and lysosomal membrane disruption

• Lysosomal enzymes leak out, become active, and digest cell components and membranes

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Distribution Mechanisms

• “Micro” Redistribution

• Acidification

• Passive Diffusion

• Blood Coagulation and

Hypostasis

• Postmortem

“Circulation”

• Putrefaction

Micro Redistribution

• Enzymes, proteases, phosphatases, glucosidases

all leak into the cytoplasm - further breaking down

cellular components

• Macromolecules, proteins, and the drugs bound to

them (or detached) drift out into the extracellular

space

• This tends to be higher in tissues rich in enzymes

(pancreas and gastric mucosa) and slower in the

heart, liver, and kidney

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Acidification

• Contents of a cell become more acidified after

death

• After a cell lyses, progressively ionized drugs

will distribute more readily as a result of being

transported in the acidic fluid in which they are

dissolved

Redistribution

40 mg/L

1 mg/L

Time Zero

Blood

Tissue

Tissue

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Redistribution

20 mg/L

10 mg/L

4 Hours Later

Blood

Tissue

Tissue

Passive Diffusion

Organs which are close to the heart and major blood

vessels

• Liver (Left Lobe)

• Stomach / Esophagus

• Adipose Tissue

• Small Intestine (Duodenum)

• Lungs

• Myocardium

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Passive Diffusion Temperature can effect this

Concentration can effect this

Blood coagulation and

hypostasis (livor)

• Blood sediments and clots unevenly after death

• This is due to blood clotting and cell lysis happening

simultaneously

• As hours pass, hypostasis occurs when the blood

sediments and serum flow, according to gravity, to

the lower parts of the body

• Drugs follow according to their respective

chemistries

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Blood coagulation and

hypostasis (livor)

Body Position

• It’s been demonstrated that body position may

influence PMR

• Repositioning of a body after death may also

influence movement of the blood postmortem

• “New” blood sources may pool near tissues

and allow more diffusion to occur

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Putrefaction

• Bacteria and microflora can effect drug

concentrations and must be considered.

• Bacteria can migrate across the intestinal wall to

blood vessels and lymph vessels

• Enteric Bacteria can metabolize drugs and produce

ethanol (as well as yeast)

• Effect can be decreased in cooler temperatures

“Postmortem Circulation”

• Rigor mortis can cause blood movement by

causing systolic pressure through ventricular

contractions

• Putrefactive processes in the abdomen can

move blood due to gas swelling

• These are not strong processes

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Ok, What can we do?

Store and Obtain Autopsy Specimens Properly

Understand the Limitations of Interpretation

Study and Review Reference Literature

Environmental Conditions

• Storing decedents between 2-8 C prior to

autopsy

• Slows redistribution

• Slows putrefaction

• Conversely, warmer temperatures have the

opposite effect

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Obtain proper autopsy

specimens

• Take blood and tissue from

specific sites during autopsy

• Central Blood (Heart, Subclavian)

• Peripheral Blood (Inferior Vena

Cava)

• Vitreous Humor

• Tissue (Liver, Brain)

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Venous Blood Collection

(Dinis-Oliveira RJ, Carvalho F, Duarte JA, et al., 1993)

Heart Blood Collection


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Liver Collection


Vitreous Humor Collection


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Testing Alternative Tissues

• Lung

• CSF

• Bone Marrow

• Skeletal Muscle


Literature Ratios

Central (Heart) / Peripheral (Venous)

Peripheral (Venous) / Tissue (Liver)

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Literature Ratios

Cocaine 1.3

Imipramine 1.8

Doxepin 5.5

Zolpidem 2.1

Reference Books

1. Basalt R, ed. Disposition of toxic drugs and

chemicals in man. 8th ed. Foster City, CA:

Biomedical Publications; 2009.

2. Osselton M, Moffat A, Widdop B, eds. Clarke's

analysis of drugs and poisons. 4th ed. Gurnee,

IL: Pharmaceutical Press; 2011. Moffat A., Osselton

M., Widdop B. and Watts J., eds.

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Examples of Drugs which exhibit

distribution – reference literature

Drug Description Vd (L/kg) Reference

Digoxin Treats atrial fibrillation 5.1-7.4 Vorphal, 1978

Morphine Analgesic 2-5 Logan, 1993

Amitripyline Tricyclic Antidepressant 6-10 Hebb, 1982

Imipramine Tricyclic Antidepressant 20-40 Jones, 1987

Ethanol Drinking Alcohol 0.43-0.59 Prouty, 1987

Diphenhydramine Antihistamine 3-14 Hargrove, 2008

Our Case

• What does our analysis show us?

• Show what the reference literature said (basalt

and article)

• Understand the clear differences

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Case – Scenario 1

Tissue Ethanol (g/100mL) Imipramine (mg/L)

Heart 0.08 14

IVC 0.09 4

Liver 0.06 (g/100g) 61 (mg/kg)

Vitreous Humor 0.11 N/A

Imipramine c/p = 3.5

Case – Scenario 2

Tissue Ethanol (g/100mL) Imipramine (mg/L)

Heart 0.02 2

IVC 0.02 0.8

Liver 0.005 (g/100g) 18 (mg/kg)

Vitreous Humor 0.03 N/A

Imipramine c/p = 2.5

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Reference Tissue Ethanol (g/100mL) Imipramine (mg/L)

Plasma N/A 0.05-0.10

Therapeutic


Blood 0.42-1.77 6-8.5

Liver 0.25-1.16 33-381


Blood 0.02-0.50 < 0.5

Liver 0.01-0.35 13

Intoxication Fatality

Natural Postmortem

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Comparison

Drug Imipramine

IVC Blood

Imipramine

Liver

Ethanol

Blood

Ethanol

Liver

Therapeutic 0.05-0.10 N/A N/A N/A

Intoxication 6-8.5 33-381 0.42-1.77 0.25-1.16

Natural <0.5 13 N/A N/A

Scenario 1 4 61 0.09 0.06

Scenario 2 0.8 18 0.02 0.005

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References 1. Bynum ND, Poklis JL, Gaffney-Kraft M, Garside D, Ropero-Miller JD. Postmortem distribution of tramadol, amitriptyline, and their metabolites in a suicidal overdose J Anal Toxicol. 2005;29(5):401-406.

2. Dinis-Oliveira RJ, Carvalho F, Duarte JA, et al. Collection of biological samples in forensic toxicology. Toxicol Mech Methods. 2010;20(7):363-414.

3. Gilliland MG, Bost RO. Alcohol in decomposed bodies: Postmortem synthesis and distribution J Forensic Sci. 1993;38(6):1266-1274.

4. Hargrove VM, McCutcheon JR. Comparison of drug concentrations taken from clamped and unclamped femoral vessels J Anal Toxicol. 2008;32(8):621-625.

5. Hebb JH,Jr, Caplan YH, Crooks CR, Mergner WJ. Blood and tissue concentrations of tricyclic antidepressant drugs in post mortem cases: Literature survey and a study of forty deaths J Anal Toxicol. 1982;6(5):209-216.

6. Hilberg T, Ripel A, Slordal L, Bjorneboe A, Morland J. The extent of postmortem drug redistribution in a rat model J Forensic Sci. 1999;44(5):956-962.

7. Jones GR, Pounder DJ. Site dependence of drug concentrations in postmortem blood--a case study J

Anal Toxicol. 1987;11(5):186-190.

8. Langford AM, Pounder DJ. Possible markers for postmortem drug redistribution. J Forensic Sci.

1997;42(1):88-92.

9. Leikin JB, Watson WA. Post-mortem toxicology: What the dead can and cannot tell us J Toxicol Clin

Toxicol. 2003;41(1):47-56.

10. Logan BK, Smirnow D. Postmortem distribution and redistribution of morphine in man. J Forensic Sci.

1996;41(2):221-229.

11. O'Sullivan JJ, McCarthy PT, Wren C. Differences in amiodarone, digoxin, flecainide and sotalol

concentrations between antemortem serum and femoral postmortem blood Hum Exp Toxicol.

1995;14(7):605-608.

References

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12. Pelissier-Alicot A-, Gaulier J-, Champsaur P, Marquet P. Mechanisms underlying postmortem redistribution of drugs: A review J Anal Toxicol. 2003;27(8):533 <last_page> 544. doi: 10.1093/jat/27.8.533.

13. Pounder DJ. The nightmare of postmortem drug changes Leg Med. 1993:163-191.

14. Pounder DJ, Jones GR. Post-mortem drug redistribution--a toxicological nightmare Forensic Sci Int. 1990;45(3):253-263.

15. Prouty RW, Anderson WH. The forensic science implications of site and temporal influences on postmortem blood-drug concentrations J Forensic Sci. 1990;35(2):243-270.

16. Prouty RW, Anderson WH. A comparison of postmortem heart blood and femoral blood ethyl alcohol concentrations J Anal Toxicol. 1987;11(5):191-197.

17. Robertson MD, Drummer OH. Postmortem distribution and redistribution of nitrobenzodiazepines in man. J Forensic Sci. 1998;43(1):9-13.

References

• 18. Robertson MD, Drummer OH. Postmortem drug metabolism by bacteria J Forensic Sci.

1995;40(3):382-386.

• 19. Vorpahl TE, Coe JI. Correlation of antemortem and postmortem digoxin levels J Forensic

Sci. 1978;23(2):329-334.

• 20. Yarema MC, Becker CE. Key concepts in postmortem drug redistribution Clin Toxicol

(Phila). 2005;43(4):235-241.

References

10/1/2014

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Thank you

84






University of Kent



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Chemistry”



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University of Kent



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Be a featured fan on an upcoming webinar! Write to us @ [email protected]

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How has ACS Webinars benefited you?

®

“providing me with resources for professional

development. In addition, I have used ACS

Webinars recordings in class or have used what I

have learned in an ACS Webinar to develop new

class materials.”

Kelly Gallagher

Associate Professor

Department of Chemistry and Biochemistry

State Univ. of New York College at Oneonta

88


@acswebinars

youtube.com/acswebinars

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89

Register for the next ACS Sci-MindTM cohort in

Identifying and Profiling Chemical Hazards*,

Nov 7 – Dec 19, 2014 and join an incredible group of

experts ready and willing to address your specific

needs. Deadline to register is October 7.

“Interested in learning more from Lucas Zarwell

on Toxicology?”

Check out what Sci-MindTM has to offer at www.sci-mind.org

Benefits of ACS Membership

90


Chemical & Engineering News (C&EN) The preeminent weekly news source.

NEW! Free Access to ACS Presentations on Demand® ACS Member only access to over 1,000 presentation recordings from recent ACS meetings and select events.

NEW! ACS Career Navigator Your source for leadership development, professional education, career services, and much more.

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