UNITED STATES DRUG TESTING
LABORATORIES, INC.
DEVELOPMENT AND UTLILITY OF
DIRECT ALCOHOL BIOMARKERS
CHARLES A. PLATE, Ph.D.
LABORATORY DIRECTOR
Testing
Innovation
Research
Development
PROPERTIES OF SELECTED
INDIRECT ALCOHOL
BIOMARKERS
INDIRECT
BIOMARKER
SENSITIVITY
SPECIFICITY
Gammaglutamyltransferase
(Blood)
61%
Alanine
aminopeptidase
activity (Urine)
77%
70%
Carbohydratedeficient transferrin
(Blood)
Ineffective(Neonates)
73% (Males)
52% (Females)
96% (Males)
94% (Females)
Testing
Innovation
Research
n/a
Development
PROPERTIES OF DIRECT
ALCOHOL BIOMARKERS
DIRECT
BIOMARKER
SENSITIVITY
SPECIFICITY
91% (EtG)
77% (EtG)
Fatty acid ethyl esters
(FAEE)
Meconium
68%
100%
Phosphatidylethanol
(PEth)
Blood
98-100%
100%
Ethyl
glucuronide/Ethyl
sulfate (EtG/EtS)
Urine
Testing
Innovation
Research
Development
STRUCTURE OF ETHYL
GLUCURONIDE
Testing
Innovation
Research
Development
STRUCTURE OF ETHYL
SULFATE
Testing
Innovation
Research
Development
SYNTHESIS OF ETHYL
GLUCURONIDE AND ETHYL SULFATE
UDP-GLUCURONYL
GLUCURONATE + EtOH
ETHYL GLUCURONATE
TRANSFERASE
SULFOTRANSFERASE
SULFATE + EtOH
Testing
ETHYL SULFATE
Innovation
Research
Development
EtG / EtS IN URINE
• Confirms alcohol exposure for up to 5 days
following consumption.
• -Glucuronidase from urinary tract infections
destroys EtG but not EtS.
• Cut-offs are variable and can be set to meet
client’s needs.
• Innocent-positives can be generated by alcoholcontaining hand sanitizers and mouthwashes.
Testing
Innovation
Research
Development
EtG ANALYSIS
EtG Cal 500 ng/ml
Testing
Innovation
Research
Development
EtS ANALYSIS
EtS Cal 125 ng/ml
125.0/80.0
Testing
Innovation
Research
Development
EtG / EtS IN URINE
• Positive EtG/EtS is NOT unequivocal evidence
of beverage alcohol consumption.
• Positive EtG/EtS requires further examination,
either clinically or by using a biomarker assay
with a higher exposure threshold for positivity.
Testing
Innovation
Research
Development
USE OF EtG / EtS IN URINE
• Determination of alcohol ingestion
– Window of measure = 1 - 5 days
– Indicates that alcohol has been consumed
– Primarily used for monitoring in enforced
abstinence programs (impaired professionals)
Testing
Innovation
Research
Development
STRUCTURE OF
PHOSPHATIDYLETHANOL
Testing
Innovation
Research
Development
SYNTHESIS OF
PHOSPHATIDYLETHANOL
PHOSPHOLIPASE D
PHOSPHATIDYLCHOLINE + EtOH
Testing
Innovation
PHOSPHATIDYLETHANOL
Research
Development
PHOSPHATIDYLETHANOL IN BLOOD
• A direct alcohol biomarker that incorporates
into cell membranes
• Long half-life--not metabolized
• Remains in red cell membrane for the life of the
blood cell or spontaneous hydrolysis - 3 weeks
• Can be detected following ingestion of 200
grams of ethanol over 1 week
• Window of detection 3 weeks or longer
Testing
Innovation
Research
Development
PHOSPHATIDYLETHANOL ANALYSIS
XIC of -MRM (3 pairs): 701.3/255.0 amu from ...
XIC of -MRM (3 pairs): 701.3/281.0 amu from ...
Max. 1.5e4 cps.
5.14
5.13
1.5e4
Max. 4.7e4 cps.
4.5e4
1.4e4
701.3/255.0
701.3/281.0
4.0e4
1.2e4
3.5e4
In te n s ity , c p s
In te n s ity , c p s
1.0e4
8000.0
6000.0
3.0e4
2.5e4
2.0e4
1.5e4
4000.0
1.0e4
2000.0
0.0
5000.0
3.67
0.0
2
4
6
8
10
Time, min
Testing
12
14
16
18
Innovation
Research
2
4
6
8
10
Time, min
Development
12
14
16
18
USE OF PHOSPHATIDYLETHANOL IN
BLOOD
• Determination of longer term alcohol abuse
–
–
–
–
Window of measure up to 3 weeks
Indicates heavy drinking over 3 week period
Identifies potential problem drinkers
Could be used to screen transplant recipients
Testing
Innovation
Research
Development
FATTY ACID ETHYL ESTER
STRUCTURE OF ETHYL OLEATE
Testing
Innovation
Research
Development
SYNTHESIS OF FATTY ACID ETHYL
ESTERS (FAEE’s)
LONG CHAIN
ACYL-CoA:ETHANOL
FATTY ACIDS + EtOH
FATTY ACID ETHYL ESTERS
O-ACYLTRANSFERASE
Testing
Innovation
Research
Development
FAEE’s IN MECONIUM
• Meconium is earliest stool of newborn
containing intestinal epithelial cells, mucus,
lanugo, amniotic fluid, bile, and water; tar-like,
sterile and odorless
• FAEE’s present in meconium of infants
delivered from known alcoholics
• Detection of FAEE’s in meconium currently
“gold standard” method of identifying infants
exposed to alcohol in utero
Testing
Innovation
Research
Development
FAEE ANALYSIS
• FAEE’s are isolated from meconium using a
solid-phase extraction technique
• FAEE’s are analyzed using positive ion (PCI)
chemical ionization gas chromatography / mass
spectrometry (GC/MS)
Testing
Innovation
Research
Development
CURRENT FAEE PROFILE
•
•
•
•
Palmitate (C16:0)
Palmitoleate (C16:1)
Stearate (C18:0)
Oleate (C18:1)
Testing
Innovation
• Linoleate (C18:2)
• Linolenate (C18:3)
• Arachidonate (C20:4)
Research
Development
FAEE’S IDENTIFY A POTENTIAL HIGH
RISK NEWBORN POPULATION
70000
62115
60000
50143
50000
40000
ng/g
30000
Hawaii
20000
Utah
10000
6628
7674
1059 1139 3133 3076
0
1st Qtr
2nd Qtr
3rd Qtr
4th Qtr
Quartiles
Testing
Innovation
Research
Development
USE OF FAEE’s IN MECONIUM
• Determination of fetal alcohol exposure in utero
–
–
–
–
Measure FAEE in fetal meconium
Window of measure > 20 weeks
Indicates alcohol usage during last half of pregnancy
Used by neonatologists when fetal alcohol exposure
suspected
Testing
Innovation
Research
Development
FUTURE POTENTIAL
APPLICATIONS FOR EtG, FAEE’s,
AND PHOSPHATIDYLETHANOL
AS DIRECT ALCOHOL
BIOMARKERS
Testing
Innovation
Research
Development
FAEE IN HAIR
• Potential biomarker for long-term alcohol
abuse (up to 3 months)
• Control group: <1 drink daily
• Patient group: 11 + drinks daily
• Hair specimens collected with interview
– 1.5 inches in length
– 100 mg in mass
– Obtained Timeline Followback for 90 days
Testing
Innovation
Research
Development
FAEE’s MEASURED
• FAEE’s separated and detected by GC/MS
–
–
–
–
–
Ethyl myristate
Ethyl palmitate
Ethyl palmitoleate
Ethyl stearate
Ethyl oleate
Testing
Innovation
(E14:0)
(E16:0)
(E16:1)
(E18:0)
(E18:1)
Research
Development
FAEE IN HAIR
ng FAEEs per gram of hair
Sum of FAEEs derived from patients and controls
8
6
4
2
Patients
Controls
0
14:00 16:00 16:01 18:00 18:01 18:02 18:03 20:04
Fatty Acids
Testing
Innovation
Research
Development
FAEE IN HAIR
Group
N
*Alcohol
(drinks/day)
Sensitivity
(%)
Specificity
(%)
Patients
25
11 ± 10
60
100
Female
6
17 ± 19
83
100
19
9±5
53
100
Male
Testing
Innovation
Research
Development
CONCLUSIONS OF FAEE IN HAIR
STUDY
• Hair FAEE’s very specific biomarkers of long
term alcohol abuse
• Sensitivity of hair FAEE’s (60%) is not
sufficiently sensitive as an assay to identify
individuals with a history of long term alcohol
abuse
Testing
Innovation
Research
Development
EtG IN HAIR
• Control group: teetotalers
• Patient group: individuals in alcohol abuse
programs
• Hair specimens collected with interview
– 1.5 inches in length
– 100 mg in mass
– Obtained Timeline Followback for 90 days
Testing
Innovation
Research
Development
EtG IN HAIR ANALYSIS
• Hair specimens washed sequentially with
hexane, methylene chloride, and methanol
• Hair specimens extracted with water
• EtG partially purified from water extracts by
solid phase extraction
• EtG resolved from water residue and identified
using LC/MS/MS
Testing
Innovation
Research
Development
HAIR EtG IN CONTROLS
AND PATIENTS
160
140
concentration pg/mg
120
100
80
60
40
20
0
0
10
20
30
40
subjects
patients
Testing
Innovation
controls
Research
Development
50
60
COMPARISON OF FAEE’s AND EtG IN
HAIR AS ALCOHOL BIOMARKERS
ALCOHOL
BIOMARKER
SENSITIVITY
(%)
SPECIFICITY
(%)
FAEE’s
60
100
EtG
80
100
Testing
Innovation
Research
Development
PATIENTS TESTING NEGATIVE FOR
HAIR FAEE’s BUT POSITIVE FOR
HAIR EtG
PATIENT
NUMBER
FAEE’S
EtG
(cut-off = 0.78)
(cut-off = 2.5)
120364
0.08
30
120376
0.18
16
123878
0.09
6
Testing
Innovation
Research
Development
CONCLUSIONS OF EtG IN HAIR
STUDY
• Hair EtG very specific biomarker of long term
alcohol abuse
• Sensitivity of hair EtG (80%) is better than any
long term marker of alcohol abuse currently
available
• Our Phase I study
– establishes feasibility of hair EtG as a long term alcohol
biomarker
– paves the way for a Phase II study to expand and
diversify the drinking population studied and validate a
hair EtG production test
Testing
Innovation
Research
Development
PHOSPHATIDYLETHANOL
• Alcohol biomarker in umbilical cord tissue
• Alcohol biomarker in newborn blood spots
• Two research studies sponsored by Phase I
SBIR grants from NIH/NIAAA
Testing
Innovation
Research
Development
PHOSPHATIDYLETHANOL IN
UMBILICAL CORD TISSUE
• Virtues of umbilical cord tissue as opposed to
meconium in drug/alcohol testing of newborns
– Easier and more dependable collection
– Greater sensitivity for certain drugs
– Availability of umbilical cord from all babies while
8- 20% of newborns lack a meconium sample due to
fetal stress
Testing
Innovation
Research
Development
PHOSPHATIDYLETHANOL IN
UMBILICAL CORD TISSUE
• FAEE’s are the direct alcohol biomarker found
in meconium; phosphatidylethanol not detected
in meconium
• Phosphatidylethanol is the direct alcohol
biomarker found in umbilical cord tissue;
FAEE’s present in umbilical cord tissue, but in
very low amounts
Testing
Innovation
Research
Development
PHOSPHATIDYLETHANOL IN
NEWBORN BLOOD SPOTS
• Newborns at high risk for fetal alcohol effects (FAE)
– Approximately 126,000 born in 2006
– Costs for medical, surgical, behavioral, custodial, and
judicial services for FAE children estimated to range
between $75 million and $9.7 billion in 2000
– Current “gold standard” alcohol biomarker test to aid in
identifying these high risk babies has a sensitivity of 68%
Testing
Innovation
Research
Development
CRITERIA THAT INITIATES TESTING OF
NEWBORNS FOR ALCOHOL OR DRUGS OF
ABUSE EXPOSURE
• Previous maternal
history of drug/alcohol
abuse
• Maternal self-report of
drug/alcohol usage
during current
pregnancy
• No prenatal care
Testing
Innovation
• No permanent address
• Presence of sexually
transmitted disease(s)
• Mother or father appear
intoxicated, “high”,
abusive, or exhibiting
inappropriate behavior
Research
Development
DO THESE CRITERIA WORK IN
IDENTIFYING DRUG/ALCOHOL EXPOSED
NEWBORNS?
• In the case of drugs of abuse YES
– Incidence of exposure in sequential births 10% or less
– Incidence when one or more criteria apply 35% or greater
• In the case of alcohol exposure NO
– Incidence of exposure in sequential births 14-18%
– Incidence when one or more criteria apply 14-18%
Testing
Innovation
Research
Development
PHOSPHATIDYLETHANOL IN
NEWBORN BLOOD SPOTS
• Potential screening test for detecting FAE
newborns with high sensitivity and specificity
• Phase I NIAAA SBIR grant to determine the
feasibility of this test was recently awarded
Testing
Innovation
Research
Development
USDTL RESEARCH FUNDING
• NIH SBIR Grants from
– National Institute of Drug Abuse
– National Institute on Alcohol Abuse and Alcoholism
Testing
Innovation
Research
Development
QUESTIONS?
Testing
Innovation
Research
Development