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development and utlility of direct alcohol biomarkers

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.

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development and utlility of direct alcohol biomarkers

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    1. DEVELOPMENT AND UTLILITY OF DIRECT ALCOHOL BIOMARKERS CHARLES A. PLATE, Ph.D. LABORATORY DIRECTOR

    6. SYNTHESIS OF ETHYL GLUCURONIDE AND ETHYL SULFATE

    7. 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.

    8. EtG ANALYSIS

    9. EtS ANALYSIS

    10. 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.

    11. 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)

    12. STRUCTURE OF PHOSPHATIDYLETHANOL

    13. SYNTHESIS OF PHOSPHATIDYLETHANOL

    14. 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

    15. PHOSPHATIDYLETHANOL ANALYSIS

    16. 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

    17. FATTY ACID ETHYL ESTER STRUCTURE OF ETHYL OLEATE

    18. SYNTHESIS OF FATTY ACID ETHYL ESTERS (FAEE’s)

    19. 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

    20. 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)

    21. CURRENT FAEE PROFILE Palmitate (C16:0) Palmitoleate (C16:1) Stearate (C18:0) Oleate (C18:1) Linoleate (C18:2) Linolenate (C18:3) Arachidonate (C20:4)

    22. FAEE’S IDENTIFY A POTENTIAL HIGH RISK NEWBORN POPULATION

    23. 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

    25. 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

    26. 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)

    27. FAEE IN HAIR

    28. FAEE IN HAIR

    29. 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

    30. 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

    31. 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

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

    34. PATIENTS TESTING NEGATIVE FOR HAIR FAEE’s BUT POSITIVE FOR HAIR EtG

    35. 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

    36. 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

    37. 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

    38. 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

    39. 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%

    40. 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

    41. 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%

    42. 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

    43. USDTL RESEARCH FUNDING NIH SBIR Grants from National Institute of Drug Abuse National Institute on Alcohol Abuse and Alcoholism

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