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)

ACUTE RECENT CHRONIC DAYS

Indirect

direct

MARKERS OF ALCOHOL ABUSE

FAEEs (EtOH + fatty acids Synthetase FAEEs)

O O C H3 CH3 O O C H3 CH3

O O CH3 C H3

O O CH3 C H3 O O CH3 C H3

O O CH3 C H3 O O CH3 C H3

ethyl palmitate ethyl palmitoleate ethyl stearate ethyl oleate ethyl linoleate ethyl linolenate ethyl arachinodate

Ethylglucuronide and Ethylsulfate

EtOH

CH3 O H OH H H H H O O H O H COOH

UDP-glucuronosyltransferase -D-ethylglucuronide (EtG) C8H14O7 (222.1945)

S OH O O O C H3

sulfotransferase ethylsulfate (EtS) C2H6O4S (126.1313)

Alcohol dehydrogenase aldehyde dehydrogenase acetic acid Kreb’s cycle

Accounting for 0.1% ingested ethanol

Placenta Fetus ABSORPTION DISTRIBUTION / METABOLISM EXCRETION

cord blood meconium

Newborn

breast milk hair

Drug

Drug/Metabolites

Blood/Plasma

Gastrointestinal Tract

faeces

Skin and Lungs

bronchial secretions/ expired air

Liver

bile

Lungs

Extra-cellular fluids and matrices: tears, nails, cerebrospinal fluid, interstitial fluid, seminal fluid, vitreous humour, pericardial fluid, saliva, sweat, hair amniotic fluid

Kidney

urine Metabolites

D/M D/M D/M D/M D/M D/M D/M D/M D/M

Urine

D/M

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 Falcón2, Simona Pichini3, Oscar Garcia- Algar4, Paolo Danesino1, Angelo Groppi1 and Aurelio Luna2

1Department of Legal Medicine, Forensic and Pharmaco-Toxicological Science, University of Pavia, Italy; 2Deparment of Legal Medicine, Universidad de Murcia, Murcia, Spain; 3Istituto Superiore di Sanità, Rome, Italy; 4Unitat de Recerca Infancia i Entorn (URIE), Institut Municipal d’Investigació Mèdica-Hospital del Mar, Barcelona, Spain.

Real samples

EtG EtS

Placenta ng/g Fetal Tissues ng/g Hair pg/g Placenta ng/g Fetal Tissues 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

• ccurs 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

• bjectively assess a

chronic exposure to drugs during fetal life, starting from second trimester of pregnancy in case of meconium whereas hair grows

• nly 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

Chan et al., JPET 2004

Fatty acid ethyl esters (FAEEs) in meconium

Positive cut-off: 7 FAEEs> 2 nmol/g meconium

Are there EtG + EtS in meconium?

2007-2009

MECONIUM SAMPLES OBTAINED FROM

Similar sociodemographic (Urban) and ethnic (Caucasians with 40% immigrants from North Africa, South America and Asia) characteristics BARCELONA, SPAIN

BCN

REGGIO EMILIA, ITALY

RE

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

* In relation to samples FAEE >2

EtS Measurable in 52% samples EtG Measurable in 84% samples

Comparison of EtG and EtS levels in meconium

1 2 3 4 5 6 7

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

Concentrations All samples (RE + BA)

EtG (nmol/g) EtS (nmol/g)*100

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

Socio-demographic characteristics

• f the different city cohorts (2)

ROMA (N= 51) REGGIO EMILIA (N= 160) CROTONE (N=96) FIRENZE (N= 99) NAPOLI (N=61) SAN DANIELE (N= 50) VERONA (N=90 ) Maternal age (years), Mean (SD) 32.3+6.3 31.9+5.53,4 29.9+5.72,4,5,6,7 34.1+5.32,3,5,7 31.9+4.53,4 32.7+5.43 32.1+4.73,4 Previous Pregnancies, Mean (SD) 1.7+1.02,3,5 0.97+1.21,4,6,7 1.3+1.01,4,6,7 1.9+1.02,3,5 0.98+1.21,4,6,7 1.8+1.12,3,5 1.7+0.92,3,5 % self-declared Teetotaler YES 11.13,4,6 NA 0.01,4,5,6,7 32.21,3,5,7 14.73,4,6 41.31,3,5,7 7.33,4,6 NO 88.83,4,6 NA 100.01,4,5,6,7 67.81,3,5,7 85.23,4,6 58.61,3,5,7 90.63,4,6 Drinking during pregnancy YES 5.52,4,5,6,7 54.21,3,4,6 7.32,4,5,6,7 40.01,2,3,7 52.01,3,6 28.01,2,3,5,7 64.21,3,4,6, NO 94.42,4,5,6,7 45.81,3,4,6 92.72,4,5,6,7 60.01,2,3,7 48.01,3,6 72.01,2,3,5,7 35.71,3,4,6, If yes, monthly 100.02,3,4,5,6,7 12.51,3,4,5,6,7 0.01,2,5,6,7 0.01,2,5,6,7 85.11,2,3,4,6 92.81,2,3,4,5 51.81,2,3,4 If yes, weekly 0.02,4,7 40.61,3,5,6 0.02,4,5,7 75.01,3,5,6 7.42,3,4,7 0.02,4,7 38.91,3,5,6 If yes, daily 0.02,4, 46.91,3,4,5,6,7 100.02 25.01,2 7.42 7.12,4 9.22 Drugs of abuse consumption YES 0.02 3.31,3,4,5,6,7§ 0.02 0.02 0.02 0.02 0.02 NO 1002 96.71,3,4,5,6,7§ 100.02 100.02 100.02 100.02 1002 §:N=30 Significantly different (p < 0.05) from: 1Roma; 2Reggio Emilia; 3Crotone; 4Firenze, 5Napoli; 6San Daniele; 7Verona

Neurodevelopmental characteristics of newborns exposed to maternal alcohol as proved by meconium screening: preliminary data

• G. Coriale, L. Tarani, S. Pichini, R. Pacifici, D.

Fiorentino, M. Fiore, F. Di Lauro, R. Marchitelli,

• G. Parlapiano, B. Scalese, M. Ceccanti

(See in the Poster Section)

n=8 children (mean age 16 months; s.d. 2.3) positive for FAEEs and EtG in meconium and n=8 children (mean age 17 months; s.d. 1.4) negative for the biomarkers in meconium were enrolled in the study. Physical growth and dysmorphological data collected by pediatrician, Cognitive motor development and the adaptive abilities assessed using the Griffiths Mental Development Scales (GMDS ) and Vineland Adaptive Behaviour Scale (VABS).

• rm

form

Results

30,0 69,5 50,8 43,5 50,9 60,9 71,6 52,5 51,3 52,5 10 20 30 40 50 60 70 80

Positive Meconium Negative Meconium Fig.1 The developmental profiles of the positiveand negative meconium sample on GMDS (Percentile)

* *

*significant comparison ( p < 0.05)

30.0 69.5 50.8 43.5 50.9 60.9 71.6 52.5 51.3 52.5 10 20 30 40 50 60 70 80

Positive Meconium Negative Meconium Fig.1 The developmental profiles of the positiveand negative meconium sample on GMDS (Percentile)

* *

*significant comparison ( p < 0.05)

Fig.1 The developmental profiles of the positiveand negative meconium sample on GMDS (Percentile)

* *

*significant comparison ( p < 0.05)

54,3 55,2 49,9 47,9 68,4 65,0 62,7 60,4 10 20 30 40 50 60 70 80

Positive Meconium Negative Meconium Figure 2 The developmental profiles of the positive and negative meconium sample

• n VABS (raw scores )

*

*significant comparison ( p < 0.05)

*

54.3 55.2 49.9 47.9 68.4 65.0 62.7 60.4 10 20 30 40 50 60 70 80

Positive Meconium Negative Meconium Figure 2 The developmental profiles of the positive and negative meconium sample

• n VABS (raw scores )

*

*significant comparison ( p < 0.05)

*

Suggested strategy to detect drinking during pregnancy:

Questionnaire and EtG in urine at first antenatal visit; if positive- EtG in hair; if positive brief intervention or detoxification centre. If suspicion, EtG in urine at each visit, standard biomarkers

Suggested strategy to detect fetal exposure to maternal drinking :

EtG in meconium for all newborns; for newborns from risky environments; for newborns of mothers suspected of drinking during pregnancy

A survey of Italian high school students regarding awareness of Fetal Alcohol Syndrome and Fetal Alcohol Spectrum Disorders

Paolo Berretta1, Roberta Dal Rin Della Mora2, Valeria Traverso2, Giacomo Toth1, Simona Pichini1, Roberta Pacifici1, Adele Minutillo1

1 Drug Abuse and Doping Unit, Department of Therapeutic

Research and Medicines Evaluation Istituto Superiore di Sanità, Roma, Italy

2 IRCCS Istituto Giannina Gaslini

0% 2% 4% 6% 8% 10% 12% 14% 16% 18% 20%

Valle d'Aosta Piemonte Lombardia Liguria Trentino Alto Adige Veneto Friuli Venezia Giulia Emilia Romagna Toscana Umbria Marche Lazio Abruzzo Molise Campania Basilicata Puglia Calabria Sardegna Sicilia

0.5% 5.5% 12.0% 2.4% 1.3% 9.9% 3.4% 5.5% 6.1% 2.4% 2.1% 5.0% 5.1% 0.5% 8.7% 2.7% 10.0% 6.3% 2.9% 7.5%

% responding schools in different regions

% Regions

• 10%

0% 10% 20% 30% 40% 50% 60% 70%

High school Technical school Vocational school 48.7% 30.5% 20.8% 47.1% 33.5% 19.3%

Distribution by type of schools

public school private school

(Question n. 8) – Does Drinking alcohol during pregnancy cause health problems to the fetus? N % No 73 0.7% Yes, if it occurs before the 12th week of pregnancy (first trimester) 383 3.9% Yes, if it occurs before the 24th week of pregnancy (second trimester) 208 2.1% Yes, at any time during pregnancy 8883 89.5% I do not know 374 3.8% Total 9921 100

Males: 4424 Females: 5497

(Question n. 9) - Does Drinking alcohol during pregnancy involve health risks? Type of school high school technical school vocational school Total N % N % N % N % No 44 0.9% 31 1.0% 23 1.1% 98 1.0% Yes, for mother health 10 0.2% 18 0.6% 17 0.8% 45 0.5% Yes, for fetus health 803 16.6% 434 14.3% 299 14.6% 1536 15.5% Yes, for the health of the two 3855 79.9% 2432 79.9% 1656 80.7% 7943 80.1% I do not know 113 2.3% 128 4.2% 58 2.8% 299 3.0% Total 4825 100 3043 100 2053 100 9921 100

(Question n. 11) If so, which alcohol amount can cause health problems of the fetus? N % A few 91 0.9% Medium 511 5.2% Much 1399 14.1% Different effects as a function of alcohol amount 7313 73.7% I do not know 607 6.1% Total 9921 100

Males: 4424 Females: 5497

(Question n.13) Which type of alcohol can be drunk during pregnancy without any problem for the fetus N % No type of spirits 5977 60.2% Wine 1338 13.5% Beer/appetizers 861 8.7% Spirits 122 1.2% All types of spirits 456 4.6% I do not know 1167 11.8% Total 9921 100

Males: 4424 Females: 5497

(Question n.13) How many standard drinks (glass of average strength of 125 ml can of beer of average strength wine 330ml glass of liquor from 40 ml) a pregnant woman can drink without causing health problems to the fetus?

• 3a. Geographic area

North Center South and islands Total N % N % N % N % None 1918 47.5% 719 46.2% 2011 46.5% 4648 46.9% Less than 3 per week 976 24.2% 360 23.1% 999 23.1% 2335 23.5% 4-5 a week 125 3.1% 44 2.8% 124 2.9% 293 3.0% 6-7 a week 26 0.6% 8 0.5% 43 1.0% 77 0.8% More than 7 per week 33 0.8% 16 1.0% 62 1.4% 111 1.1% I do not know 963 23.8% 410 26.3% 1084 25.1% 2457 24.8% Total 4041 100 1557 100 4323 100 9921 100

Available at www.iss.it/ofad/ simona.pichini@iss.it Luigi.tarani@uniroma1.it

Conclusion

The accurate assessment of fetal exposure to ethanol through the objective measurement of biomarkers could provide the basis for appropriate treatment and follow-up of exposed newborns. We are trying to develop an immunoassay for EtG in meconium, based on the same principle of that measuring EtG in adult urine and blood. This is a simple, low-cost, easy-to-perform assay (results available in less than an hour) which can be routinely applied in neonatology wards for an early diagnosis of prenatal exposure to ethanol This last is of outmost importance in order to establish an early intervention, preferably before the development of secondary disabilities. Thanks