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  • Advanced Analytics of Alcohol Consumption

    Robert D. Johnson, PhD, F-ABFT Chief Toxicologist

    Tarrant County Medical Examiner’s Office December 9-10, 2015

    Marriott City Center, Denver, CO

  • Clinical Markers for Ethanol Use

    • Fatty acid ethyl esters (FAEE’s)

    • Gamma-glutamyl transpeptidase (GGT)

    • Carbohydrate deficient transferrin (CDT)

    • Mean corpuscular volume (MCV)

  • FAEE

    • Fatty Acid Ethyl Esters (FAEEs) • Ethanol + free fatty acids = FAEEs • Used to help diagnose alcohol abuse • Concentrations increase over time as more

    alcohol is consumed

  • GGT

    • Gamma-glutamyl transpeptidase (GGT) • Used to detect liver disease • Concentrations increase over time as alcohol

    damages the liver • Good indicator of cirrhosis • Increases after 4 or more drinks/day for weeks

  • CDT

    • Carbohydrate deficient transferrin (CDT) • Used to detect recent heavy alcohol use • Normally 1-2% of transferrin is carbohydrate

    deficient • Following heavy use up to 10% can be CD • Earlier indicator than GGT

  • MCV

    • Mean corpuscular volume (MCV) • RBC volume increases after weeks of exposure to ETOH • Not sensitive enough to use alone • Useful after a period of abstinence

  • Forensic Markers for Ethanol Use

    • Ethyl glucuronide (EtG)

    • Ethyl Sulphate (EtS)

    • Serotonin metabolite ratio

  • EtG and EtS

    • Ethyl glucuronide • Ethyl sulphate

    • EtG and EtS can be detected in blood for up to 36 hrs and in urine for up to 5 days

    • Typically used to monitor abstinence in a treatment program

  • Ethyl Glucuronide

    • < 0.1% of the ethanol dose is conjugated with glucuronide (EtG)

    • Considered specific for ethanol consumption • A positive urine sample indicates alcohol

    consumption within previous 5 days

  • Ethyl Sulphate

    • < 0.05% of the ethanol dose is conjugated with sulphate (EtS)

    • Considered specific for ethanol consumption • A positive urine sample indicates alcohol

    consumption within previous 3 days

  • Serotonin Metabolite Ratio

    • 5-HTOL/5-HIAA ratio • Ethanol changes the metabolic pathway of

    serotonin • This ratio can be used to determine if alcohol

    was consumed • Ratio remains elevated for 18-24 hrs

  • OH

    N N 2H

    OH

    N H

    O

    OH

    N

    OH

    N OH

    OOH

    Serotonin

    5-HIAA

    MAO

    Aldehyde reductase

    5-HTOL

    Aldehyde dehydrogenase

    Serotonin Metabolism

    5-HIAL

    ADH

    99%1%

  • Ethanol Metabolism

    CH3CH2OH CH3CHO CH3COOH

    NAD+ NADH NADHNAD+

    ADH ALDH

    Ethanol Acetaldehyde Acetic acid

    ADH: Alcohol Dehydrogenase ALDH: Aldehyde Dehydrogenase

  • OH

    N N 2H

    OH

    N H

    O

    OH

    N

    OH

    N OH

    OOH

    Serotonin

    5-HIAA

    MAO

    Aldehyde reductase

    5-HTOL

    Aldehyde dehydrogenase

    Serotonin Metabolism

    Ethanol Consumption

    5-HIAL NADH NAD+

    NADHNAD+

    60%40%

  • Cutoff Establishment

    • Average ethanol-negative ratio: 2.52 ± 2.88

    • Average ethanol-negative ratio + 4 s.d. = 14.04 pmol/nmol.

    • Cutoff set at 15 pmol/nmol.

    • < 1 in 10,000 chance of false positive.

  • Forensic Urine Specimens a) All Data Analyzed (Ethanol Values from 0-520 mg/dL)

    0

    100

    200

    300

    400

    500

    600

    0 50 100 150 200 250 300 350 400 450 500 550 600

    - -

    5 H

    T O

    L /5

    H IA

    A R

    at io

    (p

    m ol

    /n m

    ol )

    Positives

    Negatives

    Ethanol (mg/dL)

    b) Expansion of Plot Above (Ethanol Values from 0-150 mg/dL)

    0

    20

    40

    60

    80

    100

    0 50 100 150 Ethanol (mg/dL)

    5- H

    T O

    L /5

    -H IA

    A R

    at io

    (p m

    ol /n

    m ol

    )

    Positives

    Negatives

  • Postmortem Ethanol

    • The formation of ethanol in the body after death

    • OR the formation of ethanol in a DWI blood tube following collection

  • Postmortem Ethanol

    • Approximately 100 species of bacteria, molds, and fungi form VOC’s as a part of their normal metabolism.

    • These volatiles can make an ethanol-negative sample positive

  • Cause of Postmortem Ethanol

    • Microorganisms

    - Temperature

    - Time

    - Nutrients

  • Endogenous Ethanol

    • We have billions of microbes in our bodies

    • Some of those produce ethanol (C. albicans)

    • Typically metabolized as quickly as it is produced

    • Can be measured if the instrument is sensitive enough

  • C. albicans

    • Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans

    • C. albicans is commensal and a constituent of the normal gut flora comprising microorganisms that live in the human mouth and gastrointestinal tract

    -Wikipedia

  • Volatiles Produced

    • Ethanol

    • Acetone

    • Isopropanol

    • T-Butanol

    • Acetaldehyde

    • Many others

  • Analysis of a True Ethanol Positive

    Ethanol

    Internal standard

  • Analysis of blood positive due to ethanol formation

    55 mg/dL

    Ethanol

  • Ethanol Concentration vs. Time

    0

    10

    20

    30

    40

    50

    0 24 48 72 96

    Time (hrs)

    E th

    an ol

    C on

    ce nt

    ra ti

    on (m

    g/ hg

    ) Specimen 9, NaF @ 4C Specimen 9, water @ 4C

    Specimen 9, NaF @ 25C Specimen 9, water @ 25C

  • NaF Results

    • Ethanol produced at 4°C, 25°C when tissue homogenized in water.

    • Ethanol increases up to 7500%. • Ethanol production eliminated at 4°C, 25°C

    when tissue homogenized in NaF. • Prevention of postmortem ethanol requires use

    of NaF.

    • Lewis, R.J., Johnson, R.D., Angier, M.K., Vu, N.T. Ethanol Formation in Unadulterated Postmortem Tissues. Forensic Sci. Int., 146: 17-24 (2004).

  • DWI Samples

    • DWI samples are drawn into grey-topped Vacutainer tubes

    • These contain sodium fluoride (NaF) and potassium oxalate (C2H2K2O5)

    • Whole blood is analyzed and the result is reported as g ethanol per 100 mL whole blood

  • Unknown Ethanol Origin: Case #1

    • Ohio State Trooper lost control of patrol car responding to a 911 call resulting in 3 fatalities

    • Body collected by ME, blood sample taken immediately, no ethanol detected

    • ~60 hours post-crash additional samples were collected by a contract autopsy facility

    • No preservation was used prior to specimen collection

  • Case #1 Continued

    • Urine: 70 mg/dL ethanol; Blood: 80 mg/dL. Vitreous was not available for testing

    • Media reports trooper was intoxicated at time of crash, disregarding initial negative result

    • Serotonin metabolite ratio determined to be 1 pmol/nmol proving the initial ethanol negative finding correct

  • Questions

  • Contact Info

    • Robert Johnson • rdjohnson@tarrantcounty.com • 817-920-5700 ext 8396

    Slide Number 1 Clinical Markers for Ethanol Use FAEE GGT CDT MCV Forensic Markers for Ethanol Use EtG and EtS Ethyl Glucuronide Ethyl Sulphate Serotonin Metabolite Ratio Serotonin Ethanol Metabolism Serotonin Cutoff Establishment Forensic Urine Specimens Postmortem Ethanol Postmortem Ethanol Cause of Postmortem Ethanol Endogenous Ethanol C. albicans Volatiles Produced Slide Number 23 Analysis of blood positive due to ethanol formation Slide Number 25 NaF Results DWI Samples Unknown Ethanol Origin: Case #1 Case #1 Continued Questions Contact Info

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