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Thursday, October 9, 2014
“From Molecules to Medicine: How
Structure Helps Cure Disease”
Dr. Greg Petsko, Weill Cornell Medical College
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Thursday, October 16, 2014
“Sweet Science: Having Fun with Candy
Chemistry”
Dr. Rich Hartel, University of Wisconsin-Madison
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“Chemistry of Death”
Contact ACS Webinars ® at acswebinars@acs.org
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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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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
10/1/2014
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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
10/1/2014
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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
10/1/2014
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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)
10/1/2014
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Venous Blood Collection
(Dinis-Oliveira RJ, Carvalho F, Duarte JA, et al., 1993)
Heart Blood Collection
(Dinis-Oliveira RJ, Carvalho F, Duarte JA, et al., 1993)
10/1/2014
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Liver Collection
(Dinis-Oliveira RJ, Carvalho F, Duarte JA, et al., 1993)
Vitreous Humor Collection
(Dinis-Oliveira RJ, Carvalho F, Duarte JA, et al., 1993)
10/1/2014
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Testing Alternative Tissues
• Lung
• CSF
• Bone Marrow
• Skeletal Muscle
(Dinis-Oliveira RJ, Carvalho F, Duarte JA, et al., 1993)
Literature Ratios
Central (Heart) / Peripheral (Venous)
Peripheral (Venous) / Tissue (Liver)
10/1/2014
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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.
10/1/2014
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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
10/1/2014
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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
10/1/2014
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Reference Tissue Ethanol (g/100mL) Imipramine (mg/L)
Plasma N/A 0.05-0.10
Therapeutic
Reference Tissue Ethanol (g/100mL) Imipramine (mg/L)
Blood 0.42-1.77 6-8.5
Liver 0.25-1.16 33-381
Reference Tissue Ethanol (g/100mL) Imipramine (mg/L)
Blood 0.02-0.50 < 0.5
Liver 0.01-0.35 13
Intoxication Fatality
Natural Postmortem
10/1/2014
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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
10/1/2014
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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
10/1/2014
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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
42
Thank you
84
“Chemistry of Death”
Contact ACS Webinars ® at acswebinars@acs.org
Recordings will be available to ACS members after three weeks
www.acs.org/acswebinars
Dr. Darren Griffin Professor of Genetics,
University of Kent
Lucas Zarwell Chief Toxicologist,
DC Medical Examiner's Office
10/1/2014
43
Upcoming ACS Webinars www.acs.org/acswebinars
85
®
Contact ACS Webinars ® at acswebinars@acs.org
Thursday, October 9, 2014
“From Molecules to Medicine: How
Structure Helps Cure Disease”
Dr. Greg Petsko, Weill Cornell Medical College
Dr. Martha Teeter, American Crystallographic Association
Thursday, October 16, 2014
“Sweet Science: Having Fun with Candy
Chemistry”
Dr. Rich Hartel, University of Wisconsin-Madison
Dr. Matt Hartings, American University
86
“Chemistry of Death”
Contact ACS Webinars ® at acswebinars@acs.org
Recordings will be available to ACS members after three weeks
www.acs.org/acswebinars
Dr. Darren Griffin Professor of Genetics,
University of Kent
Lucas Zarwell Chief Toxicologist,
DC Medical Examiner's Office
10/1/2014
44
Be a featured fan on an upcoming webinar! Write to us @ acswebinars@acs.org
87
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
facebook.com/acswebinars
@acswebinars
youtube.com/acswebinars
10/1/2014
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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
www.acs.org/2joinACS
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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Contact ACS Webinars ® at acswebinars@acs.org
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®
Contact ACS Webinars ® at acswebinars@acs.org
Thursday, October 9, 2014
“From Molecules to Medicine: How
Structure Helps Cure Disease”
Dr. Greg Petsko, Weill Cornell Medical College
Dr. Martha Teeter, American Crystallographic Association
Thursday, October 16, 2014
“Sweet Science: Having Fun with Candy
Chemistry”
Dr. Rich Hartel, University of Wisconsin-Madison
Dr. Matt Hartings, American University