1 NEONATAL SCREENING FOR PRENATAL ALCOHOL EXPOSURE Daphne Chan Motherisk Laboratory for Drug Exposure

2 Fetal Alcohol Spectrum Disorders Range of outcomes resulting from maternal alcohol use --> 100% preventable Incidence & cost of treatment in Canada unknown –About 1 to 3 live births per 1000 affected –Estimated $1.4 million (U.S.) per person affected Early diagnosis and intervention leads to significant improvements in development and overall quality of life –Only a small fraction of affected individuals are identified and treated Difficult to diagnosis Maternal Hx required for Dx of CNS disorders

3 Detection of Prenatal Alcohol Exposure Biological mother sometimes unavailable –Medical-legal issues Maternal self-reporting –Denial, under-reporting Maternal biomarkers –Variable sensitivity and specificity TRUE FETAL EXPOSURE Neonatal Screening Test

4 Biological markers indicative of fetal exposure Objective and independent of maternal history Ideal scenario: Hair + meconium analyses

5 ETHANOL METABOLISM ETHANOL ADH and MEOS (CYP 2E1) ACETALDEHYDE Cytosolic FAEE Synthase FAEE Non-Oxidative FATTY ACIDS Oxidative Microsomal FAEE Synthase FATTY ACYL CoA POTENTIAL BIOMARKERS

6 FATTY ACID ETHYL ESTERS (FAEE) Significant FAEE accumulation in organs and tissues commonly damaged by chronic alcoholism –Brain, heart, liver, pancreas, adipose tissue FAEE synthase activity detected in human and mouse placentae, and FAEE accumulation in mouse fetal tissues Biomarker with short and long term clinical utility –Positive blood test 24 hrs post alcohol consumption –Postmortem markers for premortem ethanol intake –Recent development of FAEE hair screening test Recently detected in the meconium of neonates exposed heavily to alcohol in utero

7 Meconium Analysis Protocol Parental Consent / Physician’s or CAS referral Collect clinical information (e.g. questionnaire, including self-reported drug use history) Review maternal and neonatal records Collect meconium sample (>1g) directly from newborn’s diaper into specimen container Extract FAEE from meconium Analyze by gas chromatography Store frozen and ship to Motherisk Lab on dry ice

8 FAEE Detected In Meconium E17 (IS) E12 E14E16 E16:1* E18 E18:1 E18:2 E20:4* E18:3* E22:6* New FAEE* included into screening profile Derived from endogenous FA or FA acquired from diet

9 Development of FAEE as Biomarkers in Meconium Selective FAEE analysis - ethyl linoleate (C18:2) [Bearer et al. 1999] FAEE spectrum analysis - profile of common esters [Moore et al. 2001] Existence of basal FAEE levels ? Positive cut-off not clearly defined ? Clinical sensitivity and specificity ?

10 Population Baseline Of Meconium Fatty Acid Ethyl Esters Among Infants Of Non-Drinking Women In Jerusalem And Toronto D. Chan; B. Bar-oz*; B. Pellerin; C. Paciorek; J. Klein; B. Kapur; D. Farine**; G. Koren. Division of Clinical Pharmacology/Toxicology, The Hospital for Sick Children, Toronto, Canada; *Department of Neonatology, Hadassah University Hospital, Jerusalem, Israel; **Department of Obstetrics, Mount Sinai Hospital, Toronto, Canada.

11 Rationale Ethanol is a metabolite of normal physiological metabolism. However, a well defined baseline and positive cut-off that accounts for the endogenous presence of FAEE does not exist for the meconium screening test in clinical practice to date.

12 Objective To determine basal FAEE levels in the meconium of neonates without prenatal alcohol exposure from 2 distinct populations Study Populations Mount Sinai Hospital (Toronto) A large urban teaching hospital that serves a culturally and ethnically diverse population Hadassah University Hospital (Jerusalem) Chosen as a negative control group as it represents a true alcohol-abstaining population because of cultural and religious reasons

13 STUDY DESIGN Jerusalem (n = 104 mothers) Toronto (n = 104 mothers) Expecting mother recruited upon admission to delivery ward Obtain Informed Consent (Verbal or Written) Questionnaire (Demographics, Diet, Drug & Alcohol Hx) Transcription of Maternal and Neonatal Health Records Meconium Sample Collection for Analysis (n = 206) Toronto (n = 102) Jerusalem (n = 104) 3 excluded due to dirty matrix 15 social drinkers excluded 84 mother-child pair included into baseline analysis 3 excluded due to dirty matrix 2 social drinkers excluded 99 mother-child pair included into baseline analysis

14 Results:

15 SUMMARY OF RESULTS FAEE species distribution was similar in Jerusalem and Toronto Social drinkers (< 1 drink per month during pregnancy) led to the accumulation of FAEE within normal baseline levels Additional presence of longer chain FAEE (E16 +) in neonates exposed to alcohol Lauric (E12), myristic (E14), and palmitic (E16:0) acid ethyl esters predominate baseline meconium

16 Important in clinical practice to distinguish between true fetal exposure and natural endogenous production Calculations of clinical sensitivity, specificity, and predictive values SENS = TP/TP + FN SPEC = TN/TN + FP + PV = TP/TP + FP - PV = TN/TN+FN DETERMINATION OF POSITIVE CUT-OFF

17 Positive cut-off was varied from the LOD (I.e. the “presence” of FAEE constituted a positive test) at intervals to 2 nmol/g (I.e. the lowest level detected from a TP case) SENS = 100%; - PV = 100% SPEC increased from 12 to 91% (+PV from 4 to 25%) Repeat calculations excluding ethyl laurate and myristate from the total FAEE sum SPEC increased from 45 to 98% (+PV from 6 to 63%) [ ]s of E12 and E14 ethyl esters in baseline and cases were insignificantly different DETERMINATION OF POSITIVE CUT-OFF

18 CONCLUSIONS FAEE exists at low levels in the meconium of neonates without prenatal alcohol exposure There is a characteristic pattern of FAEE distribution in baseline meconium (predominantly short chain FAEE), which was similarly observed in two culturally and genetically distinct populations Neonates born to minimally/ socially drinking mothers were indistinguishable from baseline Significant improvement in specificity after exclusion of ethyl laurate and myristate suggested the role of these esters in constituting the background noise

19 Future Directions Prospective study with a larger cohort of “true positives” –To verify sensitivity and specificity Development of FAEE screening test in hair Provincial/ national epidemiological study –Incidence of FASD in Canada –Prevalence of heavy drinking during pregnancy Basis for more effective public health initiatives Predictive potential of screening test? –Immediate: Correlation between laboratory result and pregnancy/ fetal outcomes –Longitudinal: Follow-up of neurobehavioral development and other social parameters

20 An alternative Harm Reduction approach to treat the mother, her child, and her future pregnancies Neonatal screening for prenatal alcohol exposure Remember…… FASD are 100% preventable