Advanced Analytics of Alcohol Consumption
Robert D. Johnson, PhD, F-ABFTChief Toxicologist
Tarrant County Medical Examiner’s OfficeDecember 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
NN 2H
OH
NH
O
OH
N
OH
NOH
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 DehydrogenaseALDH: Aldehyde Dehydrogenase
OH
NN 2H
OH
NH
O
OH
N
OH
NOH
OOH
Serotonin
5-HIAA
MAO
Aldehyde reductase
5-HTOL
Aldehyde dehydrogenase
Serotonin Metabolism
Ethanol Consumption
5-HIALNADH 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 Specimensa) 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
--
5H
TO
L/5
HIA
A R
atio
(p
mol
/nm
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 150Ethanol(mg/dL)
5-H
TO
L/5
-HIA
A R
atio
(pm
ol/n
mol
)
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 TrueEthanol 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)
Eth
anol
Con
cent
rati
on(m
g/hg
) Specimen 9, NaF @ 4CSpecimen 9, water @ 4C
Specimen 9, NaF @ 25CSpecimen 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
