prenatal exposure to ethanol Simona Pichini National Observatory on - PowerPoint PPT Presentation

Maternal-fetal biomarkers of prenatal exposure to ethanol Simona Pichini National Observatory on Alcohol, tobacco, smoking, doping and gambling Istituto Superiore di Sanità Rome, Italy

Indirect Maternal biomarkers of gestational ethanol consumption • - Interview with the mother (the most used, the most unreliable!) -Ethanol concentrations in expired air, blood and urine (very rare) - Glutamyltransferase (GGT) -Aspartate aminotransferase (AST) -Mean corpuscular volume (MCV) -N-Acetyl- β -Hexosaminidase (Beta-Hex) -apolipoprotein J -Total sialic acid -Hydroxytryptophol 5-(5-HTOL) -Phosphatidyl Ethanol (PEth) -Carbohydrate Deficient Transferrin (CDT)

MARKERS OF ALCOHOL ABUSE ACUTE RECENT CHRONIC DAYS Indirect direct

FAEEs (EtOH + fatty acids Synthetase FAEEs) O ethyl palmitate CH 3 H 3 C O O ethyl palmitoleate CH 3 H 3 C O O ethyl stearate CH 3 H 3 C O O ethyl oleate CH 3 O H 3 C O ethyl linoleate CH 3 O H 3 C O ethyl linolenate CH 3 O H 3 C O ethyl arachinodate CH 3 O H 3 C

Ethylglucuronide and Ethylsulfate Alcohol dehydrogenase Kreb’s cycle EtOH aldehyde dehydrogenase acetic acid sulfotransferase UDP-glucuronosyltransferase H COOH O O H O H 3 C H H O CH 3 S OH H O O OH O H H  -D-ethylglucuronide (EtG) ethylsulfate (EtS) C 8 H 14 O 7 (222.1945) C 2 H 6 O 4 S (126.1313) Accounting for 0.1% ingested ethanol

Drug ABSORPTION Gastrointestinal Tract Skin and Lungs Placenta D/M Metabolites DISTRIBUTION amniotic fluid Blood/Plasma Liver / METABOLISM Drug/Metabolites D/M Extra-cellular fluids and matrices: bile Fetus tears, nails, cerebrospinal fluid, interstitial fluid, seminal fluid, D/M Kidney Lungs vitreous humour, pericardial fluid, cord hair saliva, sweat, hair D/M blood D/M EXCRETION bronchial D/M D/M D/M Newborn secretions/ expired air Urine D/M faeces urine meconium D/M breast milk

Direct ethanol metabolites in maternal biological matrices • Fatty acid ethyl esters (FAEEs). • - FAEEs in blood account for acute consumption. • - FAEEs in hair account for chronic consumption as a function of hair length and segmental analysis (performed only in two labs worldwide). • Ethyl glucuronide (EtG) and Ethyl sulfate (EtS). • - EtG and EtS in blood account for recent consumption. • - EtG and EtS in urine increase the window of detection to the previous 3-4 days. • - EtG and EtS in hair account for chronic consumption as a function of hair length and segmental analysis

Biological Matrices related to gestation- delivery-neonatal period Amniotic Fluid Placenta Fetal hair Meconium Cord blood Neonatal urine

Ethyl glucuronide and ethyl sulfate in placenta and fetal tissues by LC-MS/MS: biomarkers of placental ethanol transport Luca Morini* 1 , Maria Falcón 2 , Simona Pichini 3 , Oscar Garcia- Algar 4 , Paolo Danesino 1 , Angelo Groppi 1 and Aurelio Luna 2 1 Department of Legal Medicine, Forensic and Pharmaco-Toxicological Science, University of Pavia, Italy; 2 Deparment of Legal Medicine, Universidad de Murcia, Murcia, Spain; 3 Istituto Superiore di Sanità, Rome, Italy; 4 Unitat de Recerca Infancia i Entorn (URIE), Institut Municipal d’Investigació Mèdica -Hospital del Mar, Barcelona, Spain.

Real samples EtG EtS Placenta Fetal Tissues Hair Placenta Fetal Tissues ng/g ng/g pg/g ng/g ng/g S 1-S5 <LOD <LOD <LOD <LOD <LOD S6 1305.8 391.0 59.0 125.6 50.7 S7-S16 <LOD <LOD <LOD <LOD <LOD S17 122.2 33.2 <LOD 9.6 <LOQ S18-S20 <LOD <LOD N/D <LOD <LOD S21 <LOD <LOD N/D 28.9 <LOD S22-S25 <LOD <LOD N/D <LOD <LOD S26 436.7 234.3 N/D 91.5 60.4 S27-S28 <LOD <LOD N/D <LOD <LOD S29 <LOD <LOD N/D 175.6 <LOD S30-S32 <LOD <LOD N/D <LOD <LOD S33 215.2 79.2 N/D 17.2 15.2 S34-S35 <LOD <LOD N/D <LOD <LOD SOFT/TIAFT meeting, San Francisco, CA, Sept. 2011

Similarly to amniotic fluid and placenta, cord blood objectively assess the acute exposure, which occurs at the end of the pregnancy. Cord blood is obtained during delivery and thus provides information on the last days before it. Neonatal urine is obtained the first day(s) of life and can account for the previous 60-72 hours.

As previously reported, meconium and fetal hair objectively assess a chronic exposure to drugs during fetal life, starting from second trimester of pregnancy in case of meconium whereas hair grows only during the third trimester of pregnancy.

FAEEs in neonatal hair • Caprara DL, Klein J, Koren G. Baseline measures of fatty acid etil esters in hair of neonates born to abstaining or mild social drinking mothers. Ther Drug Monit. 2005 Dec;27(6):811-5. • hair samples of infants born to women with problems of excessive alcohol consumption should be analyzed to check the possible difference in the concentrations of FAEEs • AN IMPORTANT LIMITATION IS THE AMOUNT OF NEONATAL HAIR WHICH CAN BE COLLECTED (< 10 mg) EtG and EtS in neonatal hair? • All hair samples from newborns tested < LLOQ for HEtG. Morini L, Marchei E, Vagnarelli F, Garcia Algar O, Groppi A, Mastrobattista L, Pichini S. Ethyl glucuronide and ethyl sulfate in meconium and hair-potential biomarkers of intrauterine exposure to ethanol. Forensic Sci Int. 2010 Mar 20;196(1-3):74-7. Epub 2010 Jan 8.

The first bowel movement a baby has is called meconium. Meconium is composed of amniotic fluid, mucus, lanugo (the fine hair that covers the baby's body), bile, and cells that have been shed from the skin and the intestinal tract. Meconium is thick, greenish black, sticky and sterile.

“The Meconium Project” : An Italian-Spanish Joint Study to Assess Exposure to Illicit Drugs During Pregnancy and Birth Outcomes in a mediterranean city (2004-2008) Istituto Superiore di Sanità, Roma, Italy IMIM- Hospital del Mar, Barcelona, Spain We found in 1209 meconium samples of mother-infant dyads attending the Hospital during 2002-2004 a prevalence of prenatal exposure to 2.6% Cocaine, 4.7% heroin and 5.3% cannabis and…. 45% daily maternal ethanol measured by FAEEs in meconium

Fatty acid ethyl esters (FAEEs) in meconium Positive cut-off: 7 FAEEs> 2 nmol/g meconium Chan et al., JPET 2004

Are there EtG + EtS in meconium?

2007-2009 MECONIUM SAMPLES OBTAINED FROM BCN RE BARCELONA, SPAIN REGGIO EMILIA, ITALY Similar sociodemographic (Urban) and ethnic (Caucasians with 40% immigrants from North Africa, South America and Asia) characteristics

Meconium EtG EtS Statistics Samples (nmol/g) (nmol/g) Mean 2.04 0.14 SD 3.92 0.61 FAEEs > 2 Min 0.11 0.01 nmol/g 21.94 3.47 Max (n=44) 0.21 0.02 Median EtG Measurable in 84% 0.63 0.02 Mean samples SD 1.19 0.03 FAEEs < 2 EtS Min nmol/g 0.02 1.01 Measurable (n=133) Max 7.11 0.2 in 52% samples Median 0.06 0.01 P value* 0.0002 0.009 * In relation to samples FAEE >2

Comparison of EtG and EtS levels in meconium 7 6 5 Concentrations 4 EtG (nmol/g) 3 EtS (nmol/g)*100 2 1 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93 95 97 99 All samples (RE + BA)

Receiver Operating Characteristic (ROC) curve for EtG. EtG= 2 nmol/g Based on 185 samples of newborns from teetotaler, social and problematic drinker mothers

ITALIAN MULTICENTRE STUDY 2010 Meconium samples of newborns of Neonatology Units from: Reggio Emilia- 160 Roma- 51 Napoli- 61 San Daniele del Friuli- 50 Crotone- 96 Firenze- 99 Verona- 90 All the neonates born in the Unit in a certain period of time (e.g.1 month) excluding the ones with severe pathologies (e.g. prematures, N=8) requesting intensive care

% newborns prenatally exposed to maternal ethanol in different Italian Cities: Roma- 29.4% Reggio Emilia- 10.6% Crotone- 6.2% Firenze- 5.0% Napoli- 4.9% San Daniele del Friuli- 4.0% Verona- 0% Overall: 7.9% newborns prenatally exposed to maternal ethanol