DEVELOPMENT AND UTLILITY OF DIRECT ALCOHOL BIOMARKERS CHARLES A. PLATE, Ph.D. LABORATORY DIRECTOR UNITED STATES DRUG TESTING LABORATORIES, INC. Testing

DEVELOPMENT AND UTLILITY OF DIRECT ALCOHOL BIOMARKERS

CHARLES A. PLATE, Ph.D.

LABORATORY DIRECTOR

UNITED STATES DRUG TESTING LABORATORIES, INC.

Testing Innovation Research Development


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


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


STRUCTURE OF ETHYL GLUCURONIDE


STRUCTURE OF ETHYL SULFATE


SYNTHESIS OF ETHYL GLUCURONIDE AND ETHYL SULFATE

UDP-GLUCURONYL

GLUCURONATE + EtOH ETHYL GLUCURONATE

TRANSFERASE

SULFOTRANSFERASE

SULFATE + EtOH ETHYL SULFATE


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.


EtG ANALYSIS

EtG Cal 500 ng/ml


EtS ANALYSIS

EtS Cal 125 ng/ml

125.0/80.0


• 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


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)


STRUCTURE OF PHOSPHATIDYLETHANOL


SYNTHESIS OF PHOSPHATIDYLETHANOL

PHOSPHOLIPASE D

PHOSPHATIDYLCHOLINE + EtOH PHOSPHATIDYLETHANOL


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


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


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


FATTY ACID ETHYL ESTER STRUCTURE OF ETHYL OLEATE


SYNTHESIS OF FATTY ACID ETHYL ESTERS (FAEE’s)

LONG CHAIN ACYL-CoA:ETHANOL

FATTY ACIDS + EtOH FATTY ACID ETHYL ESTERS

O-ACYLTRANSFERASE


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


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)


CURRENT FAEE PROFILE

• Palmitate (C16:0)• Palmitoleate (C16:1)• Stearate (C18:0)• Oleate (C18:1)

• Linoleate (C18:2)• Linolenate (C18:3)• Arachidonate (C20:4)


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


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


FUTURE POTENTIAL APPLICATIONS FOR EtG, FAEE’s, AND PHOSPHATIDYLETHANOL

AS DIRECT ALCOHOL BIOMARKERS


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


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)


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


FAEE IN HAIR

9 ± 5

17 ± 19

11 ± 10

*Alcohol(drinks/day)

19

6

25

N

10053Male

10083Female

10060Patients

Specificity(%)

Sensitivity(%)

Group


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


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


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


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


COMPARISON OF FAEE’s AND EtG IN HAIR AS ALCOHOL BIOMARKERS

ALCOHOLBIOMARKER SENSITIVITY

(%)SPECIFICITY

(%)

FAEE’s 60 100

EtG 80 100


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


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


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


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


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


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%


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


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%


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


USDTL RESEARCH FUNDING

• NIH SBIR Grants from

– National Institute of Drug Abuse

– National Institute on Alcohol Abuse and Alcoholism


QUESTIONS?

Documents

DEVELOPMENT AND UTLILITY OF DIRECT ALCOHOL BIOMARKERS CHARLES A. PLATE, Ph.D. LABORATORY DIRECTOR UNITED STATES DRUG TESTING LABORATORIES, INC. Testing