Motherisk Int J 2020;1:2
Gideon Koren MD, Rana Cohen BSc Pharm, MBA
Motherisk Israel Program, Adelson faculty of medicine Ariel University (2)
Address for Correspondence:
Gideon Koren MD,
Motherisk Israel, Shamir Hospital, Zrifin Israel
How can clinicians know if a baby was seriously exposed to alcohol in pregnancy? This is critical to diagnosis of fetal alcohol spectrum disorder (FASD).
Fatty acid ethyl esters (FAEE) are non- oxidative metabolites of ethanol, which accumulate in meconium of fetuses exposed to alcohol beyond the first trimester. Their presence in meconium reflects serious fetal alcohol exposure.
Identification of children suffering from FASD is often difficult, as abuse of ethanol during pregnancy is a heavily stigmatized behavior that receives little prenatal screening attention in routine care. Over the last 3 decades, measurement of the ethanol metabolites fatty acid ethyl esters (FAEE) has emerged as a useful tool to detect in the neonatal period fetal alcohol exposure starting from mid gestation.
Fetal Alcohol Spectrum Disorder (FASD) is a term used to describe a wide range of adverse physical, behavioural and cognitive effects resulting from ethanol exposure during embryonic and fetal development. The term ‘spectrum’ aims at describing arange of severity of neurodevelopmental effects The most severe manifestation of this disorder ischaracterized by a triad of pathognomonic features that include distinctive craniofacial dysmorphology (reduced palpebral fissure length, smooth filtrum, thin upper lip), intrauterine growth restriction (IUGR), and CNS developmental abnormalities (1). Unless craniofacial dysmorphology is present, which occurs relatively rarely (estimated in less than 10% of neurologically impaired individuals), diagnosis of FASD requires confirmation of maternal ethanol consumption.
The Biology of Meconium
Meconium is a fetal precursor to feces or post-natal stool, which is evacuated from the neonatal bowel over the first several days of post-natal life. The first meconium evacuation occurs within 48 hours of parturition in ninety-nine percent of neonates, with complete evacuation and transition to post-natal feces generally occurring within a maximum of 125 post-natal hours; however in very low birthweight infants, the time to passage of first meconium can be much longer ( 2).
Meconium is estimated to start depositing in the fetal intestine at approximately 12 weeks of gestation, when fetal swallowing is initiated (3). Drugs entering the fetal circulation via the umbilical cord may directly enter the meconium through the biliary system following fetal hepatic elimination, or may be deposited into the meconium through the fetal gastrointestinal system following the swallowing of drugs and/or metabolites present in the amniotic fluid, that were excreted through fetal urine )Fetal swallowing (i.e. entero-renal cycling) is considered to be the primary mechanism by which drugs concentrate in the meconium, leading to relatively high levels that are easily detected (4). Once drugs are incorporated into meconium, the matrix appears to offer a certain degree of protection from biotransformation and elimination ( 5).
FAEE in Meconium
FAEE are the products of esterification of fatty acids and ethanol, through the action of the FAEE synthase group of enzymes. Unlike the main oxidative metabolism of ethanol to acetaldehyde, the metabolic pathway of FAEE synthase is non oxidative . In the early 1990s Laposata’s group documented for the first time that FAEE are measurable in human serum after ethanol ingestion (6). In 1999 Klein et al. measured meconium FAEE in the offspring of a woman acknowledging drinking beer throughout pregnancy. The total FAEE measured by gas chromatography and flame ionization detection was 13,126 ng/g as compared to 410ng/g among control meconium samples (7).
In 2003 Bearer and colleagues validated meconium FAEE as a marker of fetal exposure to ethanol in 27 cases where maternal alcohol use was prospectively ascertained ( 8). Ethyl oleate was the FAEE most avidly correlated with reported measures of ethanol consumption. Using a cut- off level of 32ng/ml sensitivity was 84.2% and specificity 83.3% for a threshold of 1.5 average ounces of absolute alcohol ingested per drinking day.
Similar findings were documented by additional laboratories.
Similar studies have demonstrated extensive trans-placental transfer of ethylene glucuronide ( EtG), making meconium EtG a measure of both fetal and maternal metabolism of ethanol (9).
Several different compounds of FAEE have been examined in meconium, including; ethyl laurate (C14H28O2), ethyl myristate (C16H32O2), ethyl palmitate (C18H36O2), ethyl palmitoleate (C18H34O2), ethyl stearate (C20H40O2), ethyl oleate (C20H38O2), ethyl linoleate (C20H36O2), ethyl linolenate (C20H34O2), ethyl arachidonate (C22H36O2) (10 -13).
Animal studies have demonstrated strong associations between meconium FAEE concentrations and maternal gestational ethanol dose. Brien et al. reported guinea pig pup meconium FAEE concentrations 8-fold higher than non-exposed pair-fed controls with maternal (dam) ethanol dosing at a level of 4 g ethanol/kg maternal body weight/day producing mean maternal blood ethanol concentrations of 263 +/- 41 mg/dL (14). Zelner et al. reported a sensitivity and specificity of 93.3% for cumulative meconium FAEE concentrations above 0.03 nmol/g in an ovine model with a maternal ethanol dose of 0.75 g ethanol/kg maternal body weight/day producing maximum maternal and fetal blood ethanol concentrations of 0.12 +/- 0.01 g/dL and 0.11 +/- 0.01 g/dL, respectively (15).
FAEE in Meconium: Association with Adverse Outcomes
Beyond evaluation of meconium FAEE as a measure of prenatal ethanol exposure, examination of the predictive value of meconium FAEE for adverse developmental outcomes has also been reported through several animal and human studies. Brien et al. established an inverse correlation between increased meconium FAEE concentrations and brain weight in a guinea pig model of FASD (14) . Zelner et al. associated FAEE-meconium positivity with adverse renal, pulmonary, and cardiovascular endpoints consistent with the multitude of non-neurological birth defects clinically associated with prenatal ethanol exposure (15).
Elevated meconium FAEE concentrations have been associated with low birth weight (< 2500 grams), as well as decreased birth length and head circumference in clinical populations (;16-18). Noland et al. evaluated several executive functioning tasks in FAEE-meconium positive children at four years of age, identifying decreased tapping inhibition scores which identify impairments in frontal cortex functioning that are related to conflict response control (19 ). A longitudinal study evaluating mental and psychomotor development in ethanol-exposed children by the Bayley Scales of Infant Development identified associations between individual FAEE concentrations and lower mental development scores at 6.5 months (ethyl linolenate) and 2 years of age (ethyl myristate, ethyl oleate) and lower psychomotor development scores at 2 years of age (ethyl myristate, ethyl oleate, ethyl linoleate, ethyl arachidonate) (22).
Jacobson et al. reported an association between elevated ethyl oleate meconium concentrations and a diagnosis of FAS or pFAS at the age of five years. Furthermore, ethyl oleate concentrations correlated more strongly than maternal self-report with measures of recognition memory, processing speed, and complexity of symbolic play.Interestingly, the first pilot study published reporting the prospective use of meconium FAEE analysis as a neonatal screening tool identified one FAEE-positive child in a population of 30 screened infants with an uncomplicated birth and normal neonatal measures (e.g. weight, head circumference, Apgar scores, etc.), who demonstrated delays in fine and gross motor development by eight months of age and receptive language skills by fourteen months of age, requiring clinical intervention ( 23).
Use of meconium FAEE for population estimates of fetal alcohol exposure
Delano and colleagues aimed to estimate the prevalence of heavy fetal alcohol exposure in Canada through the analysis of meconium FAEEs (24 ). Meconium samples were collected in nurseries nationwide and FAEE in meconium was quantified by an established headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME GC-MS). To account for time of meconium collection after birth, data from Zelner’s research were utilized to adjust for the elevated risk of false positive results, as described earlier (23): In previous research, after serial meconium collection from the same neonates, the false positive rate was approximately 20%, 40% and 60% after 24, 48 and 72 hours, respectively . Hence a correction equation was utilized to calculate the true prevalence rates of positive meconium FAEE.
Out of 1315 samples collected in 10 Canadian obstetric units coast to coast between 2008-2011, the estimated prevalence of positive meconium FAEE ranged between 1.16% and 2.40%, translating into at least 1800 new cases of FASD in Canada each year. Positive maternal self- reports of heavy alcohol use were tenfold lower (0.24%).Approximately 32% of women reported alcohol consumption in the past 3 months in the 32-35-week gestation questionnaire.
The fact that self- reports are substantially under estimating the true rates of fetal alcohol exposure, as compared to meconium FAEE shown in this study , is consistent with a systematic review and meta- analysis, showing that the pooled prevalence of prenatal alcohol exposure was 4.26 times higher when measured by FAEE(95% CI: 1.34-13.57). This means that the majority of cases are missed without the use of an objective biomarker (25).
Alarmingly high prevalence of fetal alcohol exposure, based on positive meconium FAEE, was reported in a low socioeconomic suburb of Barcelona, Spain, at a rate of 45%. This is consistent with the reported rates of drinking in this region (26).
Other biomarkers of fetal alcohol exposure:
Ethyl glucuronide (EtG), ethyl sulfate (EtS) and fatty acid ethyl esters (FAEE) are all non-oxidative metabolites of ethanol that have been detected in meconium (27). It has been recently suggested that combined analysis of FAEE, EtS and Et G may provide a more sensitive detection of prenatal ethanol exposure, but this has not yet been demonstrated in clinical studies. FAEE currently remain the most extensively validated biomarker of prenatal ethanol exposure via meconium analysis; demonstrating a clear dose-response relationship with gestational ethanol consumption and associations with deficits in neurodevelopmental outcomes
Measurement of FAEE in hair has been developed and introduced into practice for quite some time now, since Pragst and colleagues developed the analytic methods for it (29). However, only preliminary work has been published on FAEE measurements in neonatal hair as a biomarker for in utero alcohol exposure (28-29). The major challenge is the development of sufficiently sensitive analytical methods, as levels in neonatal hair are substantially lower than in adult hair, and there is a severe limitation on the amount of hair that can be collected from neonates.
This Motherisk Update highlights the paucity of studies of biological markers for fetal exposure to ethanol. Because of the low sensitivity and specificity of maternal reports on drinking in pregnancy, due to shame, fears of prosecution and loosing custody of the child, there is a genuine need to develop and employ objective biological markers of fetal exposure to ethanol.
In addition to being used for population estimates of maternal use of alcohol, these markers can be used for clinical cases, where such measurements are critical in neonatal diagnosis.
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