The Promise of Fatty Acid Ethyl Esters and Hair Vivian Kulaga HBSc, Daniela Caprara MSc, Umar Iqbal BSc, Julia Klein MSc, Bushan Kapur PhD, James Brien PhD, James Reynold PhD, Gideon Koren MD. The Hospital for Sick Children

Fatty Acid Ethyl Esters? Oxidative Oxidative Non-oxidative FAEE (Direct biomarkers of ethanol ingestion) FAEE Synthase/Acyl-CoA: Ethanol O-Acyltransferase (AEAT)

Biomarker for Prenatal Ethanol Exposure Infants of heavy drinkers distinguished from non-drinkers by FAEE in meconium (Klein et al., 1999; Moore et al., 2003; Bearer et al., 2003; Chan et al., 2003) Infants of heavy drinkers distinguished from non-drinkers by FAEE in meconium (Klein et al., 1999; Moore et al., 2003; Bearer et al., 2003; Chan et al., 2003) FAEE detected in neonatal hair of infants born to mothers who drank socially during pregnancy (Klein et al., 2002) FAEE detected in neonatal hair of infants born to mothers who drank socially during pregnancy (Klein et al., 2002)

Guinea Pig Model- FAEE Long history as a popular animal model for FAS Long history as a popular animal model for FAS Only small mammalian born with neonatal hair Only small mammalian born with neonatal hair Recent studies show that pregnant guinea pigs treated with ethanol, and their offspring, have significantly higher levels of FAEE in the maternal hair, and neonatal hair, than controls (Brein et al, in press; Caprara et al, in press) Recent studies show that pregnant guinea pigs treated with ethanol, and their offspring, have significantly higher levels of FAEE in the maternal hair, and neonatal hair, than controls (Brein et al, in press; Caprara et al, in press)

Guinea pig testing (1) Dams exposed throughout pregnancy to 4g/d/kg ethanol, resulting in levels of 200mg% Dams exposed throughout pregnancy to 4g/d/kg ethanol, resulting in levels of 200mg% 2 control groups: ed libidum fed; sucrose replacing ethanol 2 control groups: ed libidum fed; sucrose replacing ethanol Measurement of maternal and neonatal hair Measurement of maternal and neonatal hair

Guinea pig study (2) MATERNAL HAIR SAMPLES: MATERNAL HAIR SAMPLES:.59 pmol/mg in ETOH vs.051 pmol/mg in sucrose NEONATAL HAIR SAMPLES: NEONATAL HAIR SAMPLES:.49 pmol/mg in ETOH vs.007 pmol/mg in controls

Neonatal Baseline Study 62 non-drinking or mild social drinking mothers 62 non-drinking or mild social drinking mothers Neonatal FAEE detected at very low levels in all cases (mean.3 pmol/mg) Neonatal FAEE detected at very low levels in all cases (mean.3 pmol/mg) No difference between non-drinkers and mild 3 rd trimester drinkers No difference between non-drinkers and mild 3 rd trimester drinkers

Are FAEE concentrations found in guinea pig hair reflective of those found in human hair? OBJECTIVE: To investigate if a difference in the extent of FAEE incorporation into hair exists between guinea pigs and humans, after correcting for systemic ethanol exposure

Study Design- 2˚ Analysis of exiting data from 2 published studies 1. Interventional animal study- 9 guinea pigs on high dosage regimen 9 guinea pigs on high dosage regimen (4 g/kg/day for 67 days) (Caprara et al., In press) (4 g/kg/day for 67 days) (Caprara et al., In press) Purpose/Rationale: Test validity of using FAEE as biomarker for prenatal ethanol exposure 2. Retrospective Observational Cohort study- 18 alcoholic detoxification patients (various exposures) (Wurst et al, 2004) Purpose/Rationale: Explore utility of using FAEE as long-term biomarker of alcohol use for clinical and forensic applications

Guinea pig- Peak BEC Peak BEC Vmax = 26.2 mg/dl/h Vmax = 26.2 mg/dl/h (Litvin & Switzer, 1988) FAEE measured: Ethyl myristate (E14) Ethyl myristate (E14) Ethyl palmitate (E16) Ethyl palmitate (E16) Ethyl oleate (E18:1) Ethyl oleate (E18:1) Ethyl stearate (E18) Ethyl stearate (E18) Humans- Monthly intake divided by 30 to get daily dose (g) Vmax = 24 mg/dl/h (Adachi et al., 1989) Vd = 0.59 L/Kg for women, 0.73 L/Kg for men (Marshall, et al., 1983) Weight of 58 Kg for females, and 66 Kg for males, based on known BMI, and height estimates (Grandjean, 1988 )

Systemic Exposure = AUC AUC

Correcting for Systemic Alcohol Exposure: AUC / FAEE Therefore giving you how much systemic exposure is required to produce 1 picomole of FAEE

Results (Not Normally distributed) -Significant 11-fold difference in Median AUC/FAEE

Why is there a difference? 1) Species difference in FAEE production (metabolic routes/enzymes) 2) Species difference in route/amount of deposition or incorporation (sebum/melanin) 3) Both

Conclusion When extrapolating FAEE data from hair of guinea pigs to humans, a 11-fold difference should be considered When extrapolating FAEE data from hair of guinea pigs to humans, a 11-fold difference should be considered Because guinea pigs have ~11-fold lower levels of FAEE in their hair, & still see significant levels in their pups –Good predictor that we will be able to detect significant levels in exposed neonates Because guinea pigs have ~11-fold lower levels of FAEE in their hair, & still see significant levels in their pups –Good predictor that we will be able to detect significant levels in exposed neonates

Thank you! ~ Vivian Kulaga ~ Julia Klein ~ Dr. Bushan Kapur ~ Dr. James Brien ~ Dr. Cindy Woodland ~ Daniela Caprara ~ Umar Iqbal Acknowledgements