Post on 11-Jan-2016
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DEVELOPMENT AND UTLILITY OF DIRECT ALCOHOL BIOMARKERS
CHARLES A. PLATE, Ph.D.
LABORATORY DIRECTOR
UNITED STATES DRUG TESTING LABORATORIES, INC.
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INDIRECTBIOMARKER
SENSITIVITY SPECIFICITY
Gamma-glutamyltransferase
(Blood)
61%n/a
Alanineaminopeptidaseactivity (Urine)
77% 70%
Carbohydrate-deficient transferrin
(Blood)
Ineffective(Neonates)73% (Males)
52% (Females)
96% (Males)94% (Females)
PROPERTIES OF SELECTED INDIRECT ALCOHOL
BIOMARKERS
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DIRECTBIOMARKER
SENSITIVITY SPECIFICITY
Ethylglucuronide/Ethylsulfate (EtG/EtS)
Urine
91% (EtG) 77% (EtG)
Fatty acid ethyl esters(FAEE)
Meconium
68% 100%
Phosphatidylethanol(PEth)Blood
98-100% 100%
PROPERTIES OF DIRECT ALCOHOL BIOMARKERS
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STRUCTURE OF ETHYL GLUCURONIDE
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STRUCTURE OF ETHYL SULFATE
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SYNTHESIS OF ETHYL GLUCURONIDE AND ETHYL SULFATE
UDP-GLUCURONYL
GLUCURONATE + EtOH ETHYL GLUCURONATE
TRANSFERASE
SULFOTRANSFERASE
SULFATE + EtOH ETHYL SULFATE
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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 alcohol-containing hand sanitizers and mouthwashes.
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EtG ANALYSIS
EtG Cal 500 ng/ml
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EtS ANALYSIS
EtS Cal 125 ng/ml
125.0/80.0
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• 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.
EtG / EtS IN URINE
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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)
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STRUCTURE OF PHOSPHATIDYLETHANOL
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SYNTHESIS OF PHOSPHATIDYLETHANOL
PHOSPHOLIPASE D
PHOSPHATIDYLCHOLINE + EtOH PHOSPHATIDYLETHANOL
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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
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PHOSPHATIDYLETHANOL ANALYSIS
XIC of -MRM (3 pairs): 701.3/281.0 amu from ... Max. 4.7e4 cps.
2 4 6 8 10 12 14 16 18Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
Inte
ns
ity, c
ps
5.14
XIC of -MRM (3 pairs): 701.3/255.0 amu from ... Max. 1.5e4 cps.
2 4 6 8 10 12 14 16 18Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.5e4
Inte
ns
ity, c
ps
5.13
3.67
701.3/255.0 701.3/281.0
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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
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FATTY ACID ETHYL ESTER STRUCTURE OF ETHYL OLEATE
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SYNTHESIS OF FATTY ACID ETHYL ESTERS (FAEE’s)
LONG CHAIN ACYL-CoA:ETHANOL
FATTY ACIDS + EtOH FATTY ACID ETHYL ESTERS
O-ACYLTRANSFERASE
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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
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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)
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CURRENT FAEE PROFILE
• Palmitate (C16:0)• Palmitoleate (C16:1)• Stearate (C18:0)• Oleate (C18:1)
• Linoleate (C18:2)• Linolenate (C18:3)• Arachidonate (C20:4)
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FAEE’S IDENTIFY A POTENTIAL HIGH RISK NEWBORN POPULATION
10591139 3133 307666287674
62115
50143
0
10000
20000
30000
40000
50000
60000
70000
ng/g
1st Qtr 2nd Qtr 3rd Qtr 4th Qtr
Quartiles
Hawaii
Utah
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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
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FUTURE POTENTIAL APPLICATIONS FOR EtG, FAEE’s, AND PHOSPHATIDYLETHANOL
AS DIRECT ALCOHOL BIOMARKERS
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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
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FAEE’s MEASURED
• FAEE’s separated and detected by GC/MS– Ethyl myristate (E14:0)
– Ethyl palmitate (E16:0)
– Ethyl palmitoleate (E16:1)
– Ethyl stearate (E18:0)
– Ethyl oleate (E18:1)
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FAEE IN HAIR
Sum of FAEEs derived from patients and controls
0
2
4
6
8
14:00 16:00 16:01 18:00 18:01 18:02 18:03 20:04
Fatty Acids
ng
FA
EE
s p
er g
ram
of
hai
r
Patients
Controls
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FAEE IN HAIR
9 ± 5
17 ± 19
11 ± 10
*Alcohol(drinks/day)
19
6
25
N
10053Male
10083Female
10060Patients
Specificity(%)
Sensitivity(%)
Group
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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
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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
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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
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HAIR EtG IN CONTROLS AND PATIENTS
0
20
40
60
80
100
120
140
160
0 10 20 30 40 50 60
subjects
con
cen
trat
ion
pg
/mg
patients controls
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COMPARISON OF FAEE’s AND EtG IN HAIR AS ALCOHOL BIOMARKERS
ALCOHOLBIOMARKER SENSITIVITY
(%)SPECIFICITY
(%)
FAEE’s 60 100
EtG 80 100
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PATIENTS TESTING NEGATIVE FOR HAIR FAEE’s BUT POSITIVE FOR
HAIR EtG
PATIENTNUMBER
FAEE’S(cut-off = 0.78)
EtG(cut-off = 2.5)
120364 0.08 30
120376 0.18 16
123878 0.09 6
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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
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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
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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
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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
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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%
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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
• No permanent address
• Presence of sexually transmitted disease(s)
• Mother or father appear intoxicated, “high”, abusive, or exhibiting inappropriate behavior
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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%
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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
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USDTL RESEARCH FUNDING
• NIH SBIR Grants from
– National Institute of Drug Abuse
– National Institute on Alcohol Abuse and Alcoholism
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QUESTIONS?