DEVELOPMENT AND UTLILITY OF DIRECT ALCOHOL BIOMARKERS 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 Testing Innovation Research Development

PROPERTIES OF DIRECT ALCOHOL BIOMARKERS 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 ETHYL SULFATE Testing 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 alcohol-containing 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 PHOSPHATIDYLETHANOL Testing Innovation 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 701.3/255.0 701.3/281.0 Testing Innovation Research Development

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 Linoleate (C18:2) Palmitate (C16:0) Linolenate (C18:3) Arachidonate (C20:4) Palmitate (C16:0) Palmitoleate (C16:1) Stearate (C18:0) Oleate (C18:1) Testing Innovation Research Development

FAEE’S IDENTIFY A POTENTIAL HIGH RISK NEWBORN POPULATION 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 (E14:0) Ethyl palmitate (E16:0) Ethyl palmitoleate (E16:1) Ethyl stearate (E18:0) Ethyl oleate (E18:1) Testing Innovation Research Development

FAEE IN HAIR Testing Innovation Research Development

FAEE IN HAIR Group N *Alcohol Sensitivity Specificity (drinks/day) (%) (%) Patients 25 11 ± 10 60 100 Female 6 17 ± 19 83 100 Male 19 9 ± 5 53 100 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 Testing Innovation Research Development

COMPARISON OF FAEE’s AND EtG IN HAIR AS ALCOHOL BIOMARKERS Testing Innovation Research Development

PATIENTS TESTING NEGATIVE FOR HAIR FAEE’s BUT POSITIVE FOR HAIR EtG 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 No permanent address Presence of sexually transmitted disease(s) Mother or father appear intoxicated, “high”, abusive, or exhibiting inappropriate behavior Previous maternal history of drug/alcohol abuse Maternal self-report of drug/alcohol usage during current pregnancy No prenatal care Testing Innovation 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