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Journal of Neonatal-Perinatal Medicine 7 (2014) 47–54 DOI 10.3233/NPM-1471413 IOS Press
Original Research
Prenatal alcohol exposure and its repercussion on newborns A. Ba˜naa , M.J. Tabernerob , A. P´erez-Mu˜nuzuria , O. L´opez-Su´areza , S. Dosila , P. Cabarcosb , A. Bermejob , J.M. Fragaa and M.L. Coucea,∗ a Servicio
de Neonatolog´ıa, Departamento de Pediatria, Hospital Cl´ınico Universitario de Santiago, Santiago de Compostela, Spain b Departamento de Medicina Legal, Facultad de Medicina, Universidad de Santiago, Santiago de Compostela, Spain
Received 21 March 2013 Revised 27 November 2013 Accepted 14 January 2014
Abstract. BACKGROUND: Alcohol consumption during pregnancy, even when moderate, implies a risk of impaired neurodevelopment, physical impairments and malformations. Its early identification is essential for establishing preventive measures to diminish disabilities among newborns. METHODS: To determine the frequency of consumption of substance use in pregnant women, we have used the techniques of gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry to detect drugs and markers of chronic consumption of alcohol in meconium. We performed a prospective study during a period of 10 months among 110 infants in our hospital, assessing anthropometry, neuromuscular development and determination of toxic substances in urine and meconium. Furthermore, meconium analysis identified fatty acid ethyl esters (FAEEs) and ethyl glucuronide (Etg). We also conducted a survey regarding the obstetric history, toxic habits, and employment status of the mothers. RESULTS: According to early detection markers analyzed in meconium (FAEE >1000 ng/g and/or Etg >50 ng/g meconium), 34.65% of pregnant women consumed alcohol during pregnancy, and 17% were positive for both markers. Within the positive cases, 50% of those exceeding a FAEE’s value of 5000 ng/g in meconium had low birth-weight children. Only 5/110 mothers (4.5%) admitted to occasional alcohol consumption during pregnancy. Nobody admitted to frequent intake. The cocaine test was positive in three cases; two of them were positive for alcohol as well. CONCLUSION: As expected, many screening devices do not accurately capture use during pregnancy and supplemental methods such as meconium analysis of biomarkers of chronic alcohol consumption may be warranted. Keywords: Alcohol consumption, pregnancy, meconium, faees, ethyl glucuronide
1. Introduction
∗ Corresponding
author: Dr. M.L. Couce, Departamento de Pediatria, Hospital Cl´ınico Universit´ario de Santiago de Compostela, A Choupana, s/n◦ . 15706 Santiago de Compostela, Spain. Tel.: +34 981 950 167; E-mail: [email protected].
Investigation of alcohol consumption in pregnant women is of great importance, as this consumption is the third cause of childhood intellectual disability in countries with drinking traditions, as well as the cause that is 100% preventable [1, 2]. Alcohol consumption
1934-5798/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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is deeply rooted in Western culture, and Spain is one of the world’s major producers and consumers of alcohol. Alcohol consumption during pregnancy may lead to fetal damage involving neurodevelopment abnormalities and intellectual disability. These alterations are named Fetal Alcohol Spectrum Disorders (FASD), which describes a continuum of permanent birth defects caused by maternal consumption of alcohol during pregnancy, which includes, but is not limited to fetal alcohol syndrome (FAS) [3, 4]. This fact implies great difficulty for clinical diagnosis. In addition it is not easy to identify mothers who consumed alcohol during pregnancy because they usually deny it [5, 6]. There are biological markers of chronic consumption such as ethyl glucuronide (Etg) [7, 8] and the ethyl esters of fatty acids (FAEE) [9, 10], which would allow (with laboratory analysis) the identification and early intervention of affected children. The main importance of those chronic consumption markers lies in their wide window of detection; i.e. they remain in the biological samples for much longer than the original compound (in this case, ethanol), giving more scope for the analysis and the detection of consumption. Those biological markers would also allow identifying the low-to-moderate consumption of alcohol during pregnancy, thereby detecting a group of neonates that could be at risk of poor neurodevelopment [1, 11, 12]. At the present time, there is no single laboratory marker capable of demonstrating consumption of alcohol with certainty. Many clinical trials show that an early identification of children exposed to alcohol and/or drug abuse during pregnancy diminishes the disabilities of those children [13–15]. Xenobiotics consumed by women during pregnancy can be identified in the meconium and hair of the newborn [9, 16]. Because of its volatility and its potential absorption from external sources, it is not possible to determine ethanol in hair. Meconium is a fundamental matrix, as it represents a deposit of all agents that the baby has been exposed to during the last trimester of gestation, because it forms after the 12th week [17, 18]. However, it must be taken into account that meconium is a very complex matrix, containing very low concentrations of analytes, and requires previous purification of the sample and sensitive analytic techniques. The objective of this work is to know the prevalence of the positive biomarkers of alcohol in newborns with
prenatal alcohol exposure in our area (covers urban and rural area), as well as establishing the analytic procedures to identify the Etg and the FAEEs in the newborn’s meconium as a routine practice, this practice is not standardized in this moment.
2. Method 2.1. Study population We performed a prospective study during a 10 month period (February–December 2010) in neonates born in the maternity ward of the Hospital Cl´ınico Universitario of Santiago de Compostela. This hospital is a tertiary referral care center for a population of 394,172 people (including rural and urban areas), and has a level 3b Neonatal Unit. During this period 2,594 babies were born. Inclusion criteria were: neonates ≥32 weeks of gestation and/or ≥1500 g weight at birth and belonging to our health area. Exclusion criteria were: babies affected from major malformations or severe diseases, or consent refusal from the parents. Inclusion criteria were met by 2,466 patients. Three babies per day were selected at random, one day each week, during the morning shift. Of these 129 newborns, 19 parents refused consent; thus, finally, 110 babies were included in the study. 2.2. Maternal data The Hospital’s Ethics Committee approved the study, and mothers were asked for informed consent. Study staff gave a survey to the mothers, and the responses were seen by the study staff during the collection process. Questionnaire data was obtained about: mothers’ age, level of education (primary, secondary, university), employment status (stable, unemployed, housewife), drug consumption (alcohol and/or other drugs), over-the-counter medications or mouthwash which contain alcohol, perinatal and obstetric antecedents (gestational controls, previous abortions). Alcohol consumption was evaluated by the system of standard drinking units [19], which sums the number of glasses of wine (∼10 g), bottles of beer (∼10 g) and units of spirits (∼10 g) consumed regularly per week. We consider occasional consumption of alcohol to be 1–2 drinks per week containing 10 g of pure alcohol content each. Habitual consumption would be above this quantity.
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2.3. Newborn data Several data were evaluated for each newborn, such as: gestational age, Apgar score, anthropometric parameters (length, weight, head circumference), neuromuscular evaluation (Ballard’s test). Samples of the first urine and meconium from each newborn were also collected. 2.4. Perinatal levels of drug exposure Urine samples of newborns were analyzed by a quick drug test (screening done by immunoassay) that could alert us of recent drug consumption, such as: cocaine, methadone, opiates, benzodiazepines, cannabis and amphetamine compounds. Positive cases were confirmed by gas chromatography/mass spectrometry (GC/MS). The detection of FAEEs and Etg in meconium was performed by gas chromatography / mass spectrometry (GC/MS) and by liquid chromatography/tandem mass spectrometry (LC/MS/MS) because of the sensitivity and specificity of those techniques, since meconium is a biological sample where the concentration of analytes is very low. The amount of meconium used was 500 mg for each analysis. An analytic method with LC/MS/MS was developed to determinate the presence of Etg in meconium by a linear calibration line at range 50–200 ng/g, where the LOD was 20 ng/g, and the LOQ was 50 ng/g. A deuterated internal standard (ethyl glucuronide d5 ) was used for quantification. After a sample pretreatment with Acetonitrile, we used solid phase (Bond Elut Certify columns) for the extraction procedure. The development of the analytic methodology needed for the determination of FAEEs in meconium, was performed using GC/MS. As extraction technique, we opted for MicrowaveAssisted Extraction (MAE), because it was the most efficient extractive technique and the one that obtained the cleanest extracts. MAE has scarcely been used to extract drugs of abuse from different materials such as serum, urine, tablets and coca leaves. Several authors have shown the extraction capacity and advantages of the MAE [20–23]. The analytical method for Etg and FAEEs was completely validated regarding linearity, limits of detection and quantification, repeatability (intraday precision), reproducibility (interday precision) and recovery, following the recommendations of the FDA [24].
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Both markers (FAEEs and Etg) were determined when there was sufficient amount of the biological sample available, since it had to be previously divided for the two determinations (this was done, as mentioned above, with different techniques of extraction and analysis). When this was not possible, only one of the markers was chosen for the analysis, depending on the availability of the equipments. The results were considered positive if the FAEE’s value was >1000 ng/g meconium, and if the Etg’s value was >50 ng/g meconium. In the literature there are no clearly defined criteria about the concentration that these markers need to reach in meconium to be considered as positive [8, 25, 26]. In the literature, FAEE concentration of 2 nmol/g (the equivalent to 600 ng/g) is the value established as cut-off for prenatal exposure to alcohol, to ensure that there has been alcohol consumption during pregnancy [8, 25]. We chose a value of 1000 ng/g to avoid false positive results due to endogen FAEEs, as it is known that ethyl myristate is positive in 80% of meconium samples of non-exposed babies [27]. Regarding ethyl glucuronide, there are also different criteria [28, 29]. In our study, such as in Tarcomnicu’s [30], we considered a positive value starting at a concentration of 50 ng/g. The analytical technique used for this immunologic screening of urine to detect drugs of abuse was a COBAS INTEGRA 400® 2.5. Data analysis A descriptive analysis was performed using the SPSS 15.0 programme for Windows. Data are expressed as mean ± SD or as absolute frequencies and percentiles. A linear regression analysis was performed to correlate alcohol consumption and birth weight. Values of p < 0.05 were considered statistically significant.
3. Results One hundred and one valid meconium samples were obtained from the newborns included in the study. Both Etg and FAEEs were determined in 47 cases; whereas, FAEEs or Etg alone, were determined in 35 and 19 cases, respectively. The decision of determining one or both biochemical markers was made according to the amount of sample available. The result was positive for one or two of the markers in 35 cases, which means a frequency of 34.65% of the babies studied; in
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A. Ba˜na et al. / Prenatal alcohol exposure Table 1 Etg and FAEE values on meconium in patients with at least one positive result
Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Ethyl-glucuronide (ng/g meconium)
Fatty acids ethyl esters (ng/g meconium)
No 56 No (–) 86 No 189 No No 63 55 74 218 No (–) No (–) No No No No (–) (–) (–) (–) 73 (–) (–) 95 263 (–) (–) (–) (–) No
5500.30 144.35 1352.30 17462 546.86 17994 7079.70 5377.40 1925 296.54 169.15 1299.10 4311.20 1417 4260.60 1399.50 8969.10 3286.50 1600.80 112427 3039.50 1225.40 99116 7006.50 3543 No 1492.30 2181.30 44470 No 1004.10 2715.60 1881.70 27892 45533
No: not detected; Negative: (–).
8 cases (17%) both markers were positive (Table 1). An overview of the cumulative FAEEs concentrations and Etg concentrations is given in Table 2. Urine quick drug test was performed in all the cases. Three of the urine samples gave a positive result for cocaine, and all three were confirmed by GC/MS. Two of these cases were also positive for alcohol (n 1, 4) without evidence of abuse of other drugs. These positive results were later confirmed with the meconium samples. The method used to obtain this confirmation is the same used in a study published by some of the authors of the present study [31]. None of the newborns included in the study showed remarkable neurological disorders, and the Ballard’s test was concordant to their morphological age.
Table 2 Cumulative fatty acid ethyl esters (FAEEs) and ethyl glucuronide (Etg) concentrations in the 110 meconium samples analyzed Cumulative concentration (FAEEs) (ng/g) (LOD = 100 ng/g) (LOQ = 1000 ng/g) 5001 Etg concentration (ng/g) (LOD = 20 ng/g) (LOQ = 50 ng/g) 50
Number of samples 4 48 18 12
5 13 10
Regarding anthropometric parameters, there were no significant differences between the group of babies exposed to alcohol and the non-exposed; however, a correlation between alcohol consumption and low birth weight was observed, since 20% of the patients that showed a positive result presented low birth weight. This correlation is even greater if we take into account those patients with values of meconium FAEEs over 10000 ng/g meconium (3, 4) (Table 3). However, on a regression analysis after adjusting for potential confounders such as gestational age (p < 0.001), mothers’ smoking habits (p = 0.274) and sex of the newborn (p = 0.070), no statistically significant correlation between markers of alcohol consumption and birth weight was found. Twenty three of the 110 patients studied required admittance to the Neonatal Unit; of which 8 were admitted to the Neonatal Intensive Care Unit. The main causes were: prematurity of 32–35 weeks (7), respiratory distress (6), children of insulin-dependent diabetic mothers (3), congenital malformations (3), and other causes (4). There was no significant association between alcohol exposure and need of admittance to the Neonatal Unit. The 3 patients admitted with a positive result for cocaine showed clinical data that justified the toxicological analysis. One of them was the son of a mother with self-admitted consumption of cocaine during gestation; another one presented intense jitteriness from birth, and the third one was a polimalformative syndrome, associated with cocaine abuse. None of them developed neonatal withdrawal. Regarding the mothers, 91.42% of the mothers with a baby showing a positive result (above the cut-off for Etg or for cumulative FAEEs) denied any consumption and only 5 (4.5%) admitted occasional alcohol consumption during pregnancy. No one admitted frequent
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Table 3 Fatty acid ethyl esters (FAEEs) and birthweigth Weight AGA SGA
10000 ng/g meconium
67 13
13 1
4 1
4 3
Spearman’s r = 0.15 (p = 0.182). AGA: weight is appropriate for gestational age; SGA: weight is small for gestational age. Table 4 Maternal features as markers of alcohol screening in children Maternal features
Children with positive alcohol markers (n. 35)
Children with negative alcohol markers (n. 75)
Statistical significance
Age Level of education
31.90 ± 4.4 PE: 14 (40%) SE: 13(37%) UE: 8 (23%) W: 22(63%) U: 6(17%) H: 7(20%) 3 (8.6%) 0 35 (100%) 7 (20%)
32.54 ± 3.7 PE: 16 (21%) SE: 48 (64%) UE: 11(15%) W: 57 (76%) U: 12 (16%) H: 6 (8%) 2 (2.6%) 1 74 (98.6%) 16 (21.3%)
P = 0.89 P = 0.83
Work status
Admission of alcohol’s consumption Admission of illicit drug consumption Controlled Pregnancy Previous abortions
P = 0.7
NA NA P = 0.25 P = 0.382
PE: primary education; SE: secondary education; UE: university education; W: working; U: unemployed; H: housewife. NA: Not applicable.
consumption. None claimed exposure to other sources of alcohol such as over-the-counter medications or mouthwash containing alcohol. Currently, the alcohol content of these external sources is insignificant; thus, the possibility that such minor metabolites of alcohol as Etg or FAEEs reach the meconium is not taken into account. There was no significant association between alcohol consumption and mother’s age (average age of 31.9 ± 4.4 for mothers whose babies showed positive alcohol markers, versus 32.54 ± 3.7 for mothers whose babies showed negative alcohol markers. In mothers under 30 years of age, the markers were positive 24.32% of cases). There was no significant association either between alcohol consumption and level of education or employment status of the mothers. All of the pregnancies were supervised by the midwife/obstetrician (Table 4).
4. Discussion We noticed a high percentage of pregnant women (34.6%) who ingested alcohol during pregnancy as it was reflected by the early detection markers analyzed in their children and confirms other literature finding
that many women do not admit to substance use during pregnancy, as reason that the biomarker approach is valuable. Garc´ıa-Algar [32] already alerted in 2008 about the high prevalence of fetal alcohol exposure (45%) in their study carried out in a low socioeconomic status cohort of a Mediterranean city (Barcelona). Another assay performed in several cities of Italy, shows a considerable variability in the prevalence of fetal exposure to ethanol, reaching a figure of 29.4% in cities like Rome [25]. These data, together with data that we have obtained, indicate that gestational ethanol exposure is widespread, at least in parts of Southern Europe. In a survey carried out among Italian and Spanish neonatologists regarding the awareness of detection of FAS and FASD during pregnancy, approximately 50% of the Italian neonatologists and 40% of the Spanish neonatologists allowed women to drink an occasional glass of wine or beer during pregnancy [33]; it should be emphasaized that small amount of alcohol is dangerous and can lead to neurodevelopmental deficits. In addition to these high percentages, we must add the fact that mothers don’t usually admit to consumption of alcohol or other drugs due to social stigmas associated with such consumption (91.9% of the mothers with a positive result for alcohol consumption in
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our cohort didn’t admit any kind of toxic abuse during pregnancy). Anyway we do not used a validated test and several screening tools have been validated for use in prenatal care settings, and the most recently proposed brief and easy to use T-ACER3 has demonstrated high sensitivity and specificity in both identifying risk drinking during pregnancy [34]. These last two findings would justify there are not many references regarding the neurodevelopment repercussion of a moderate prenatal exposure to alcohol, FASD (which in younger populations of the USA and some countries of Western Europe might reach to 2–5%) [4]. All this would show the importance of developing methods be a valid supplement to oral screening instruments for analyzing biological markers and allow the early detection of those children at risk of deficient neurodevelopment. This knowledge would permit an early intervention, which would lead to improvement of executive function and emotional problem-solving skills [11, 35]. In our study we determined ethyl glucuronide and/or ethyl esters of fatty acids. These markers, specifically FAEES are validated biomarkers of fetal alcohol exposure [6] but do not permit an accurate quantitative assessment (the quantification criteria are not standardized and they depend on each author’s opinion and of the FAEEs chosen for analysis) but they allow establishing a correlation with alcohol consumption as well as differentiating between occasional consumption and chronic or abusive consumption [7, 8, 16]. Other markers could be of interest, such as phosphathydilethanol [36] and carbohydrate-deficient transferring (CDT) [37]. The determination of phosphathydilethanol in blood is useful because of its wide window of detection (1–3 weeks of abstinence) and it is a feasible procedure [38]. The determination of carbohydrate-deficient transferring (CDT) is useful because it is a surrogate indirect marker that increases in blood when there is chronic alcohol consumption. A worldwide change in drinking habits is being confirmed, and there is a higher consumption among groups within the higher cultural and economic levels, as well as an increase in consumption in women between the ages of 16–24 [39]. In our study, as a consequence of the general tendency to delay the first pregnancy (mean maternal age was 31.4), we didn’t observe a greater consumption among younger mothers. Instead, we did observe an increase in consumption among housewives and university-educated women. Those results are in disagreement with the Pichini
study, where low maternal education was associated with biomarker scores over the cutoff [25]. We observed, as in other studies [40, 41], a correlation between alcohol exposure and low birth weight, present in up to 42.8% of those who exceed a value of FAEE of 5000 ng/g in meconium. The neurological exams didn’t detect assessable abnormalities in the immediate perinatal period, but a further follow up is needed set up strategies for functional assessment, individualized planning, structured teaching, and developments in cognitive-behavioral methods of interventions for those identified to detect some cognitive impairments that could be treated early and thus improve those patients’ evolution as far as possible. We consider that further studies are needed to asses this potential long-term repercussion. Regarding the consumption of others drugs of abuse, analytical determinations in meconium showed that three mothers had consumed cocaine, and two of them had also consumed alcohol. This data agree with the EMCDDA annual report of 2011, which points to Spain as the country with the highest cocaine consumption. Prenatal exposure to cocaine can produce neurotoxic effects and malformations as a result of blood flow interruption and due to its teratogenic action [42, 43]. Two of our three patients with fetal exposure to cocaine showed these alterations probably caused by such exposure. Observed findings indicate that fetal exposure to alcohol has such a high detrimental impact, that it would be worthwhile to make all the necessary efforts to establish routine early diagnosis of the problem.
5. Conclusions Small or moderate alcohol consumption during pregnancy is underestimated. Thus, there is a group of children at risk of suffering neurodevelopment disorders. These children could be benefited by early stimulation and follow up. Ethyl glucuronide and ethyl esters of fatty acids are useful biomarkers that allow detection of alcohol consumption during pregnancy; thus, their use could be standardized. We still cannot determine with certainty which is the best marker, so we propose the use of both, and even of some others such as CDT or phosphatydilethanol, because the presence of endogenous FAEEs may lead to false positive cases, and Etg’s levels are sometimes
A. Ba˜na et al. / Prenatal alcohol exposure
so low that they cannot be detected. As a consequence, we agree with other authors regarding the importance of the analysis of several alcohol consumption markers, because the results obtained using various markers are more reliable.
[14]
[15]
[16]
Financial disclosure The authors have nothing to disclose. [17]
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Journal of Neonatal-Perinatal Medicine 7 (2014) 47–54 DOI 10.3233/NPM-1471413 IOS Press
Original Research
Prenatal alcohol exposure and its repercussion on newborns A. Ba˜naa , M.J. Tabernerob , A. P´erez-Mu˜nuzuria , O. L´opez-Su´areza , S. Dosila , P. Cabarcosb , A. Bermejob , J.M. Fragaa and M.L. Coucea,∗ a Servicio
de Neonatolog´ıa, Departamento de Pediatria, Hospital Cl´ınico Universitario de Santiago, Santiago de Compostela, Spain b Departamento de Medicina Legal, Facultad de Medicina, Universidad de Santiago, Santiago de Compostela, Spain
Received 21 March 2013 Revised 27 November 2013 Accepted 14 January 2014
Abstract. BACKGROUND: Alcohol consumption during pregnancy, even when moderate, implies a risk of impaired neurodevelopment, physical impairments and malformations. Its early identification is essential for establishing preventive measures to diminish disabilities among newborns. METHODS: To determine the frequency of consumption of substance use in pregnant women, we have used the techniques of gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry to detect drugs and markers of chronic consumption of alcohol in meconium. We performed a prospective study during a period of 10 months among 110 infants in our hospital, assessing anthropometry, neuromuscular development and determination of toxic substances in urine and meconium. Furthermore, meconium analysis identified fatty acid ethyl esters (FAEEs) and ethyl glucuronide (Etg). We also conducted a survey regarding the obstetric history, toxic habits, and employment status of the mothers. RESULTS: According to early detection markers analyzed in meconium (FAEE >1000 ng/g and/or Etg >50 ng/g meconium), 34.65% of pregnant women consumed alcohol during pregnancy, and 17% were positive for both markers. Within the positive cases, 50% of those exceeding a FAEE’s value of 5000 ng/g in meconium had low birth-weight children. Only 5/110 mothers (4.5%) admitted to occasional alcohol consumption during pregnancy. Nobody admitted to frequent intake. The cocaine test was positive in three cases; two of them were positive for alcohol as well. CONCLUSION: As expected, many screening devices do not accurately capture use during pregnancy and supplemental methods such as meconium analysis of biomarkers of chronic alcohol consumption may be warranted. Keywords: Alcohol consumption, pregnancy, meconium, faees, ethyl glucuronide
1. Introduction
∗ Corresponding
author: Dr. M.L. Couce, Departamento de Pediatria, Hospital Cl´ınico Universit´ario de Santiago de Compostela, A Choupana, s/n◦ . 15706 Santiago de Compostela, Spain. Tel.: +34 981 950 167; E-mail: [email protected].
Investigation of alcohol consumption in pregnant women is of great importance, as this consumption is the third cause of childhood intellectual disability in countries with drinking traditions, as well as the cause that is 100% preventable [1, 2]. Alcohol consumption
1934-5798/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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is deeply rooted in Western culture, and Spain is one of the world’s major producers and consumers of alcohol. Alcohol consumption during pregnancy may lead to fetal damage involving neurodevelopment abnormalities and intellectual disability. These alterations are named Fetal Alcohol Spectrum Disorders (FASD), which describes a continuum of permanent birth defects caused by maternal consumption of alcohol during pregnancy, which includes, but is not limited to fetal alcohol syndrome (FAS) [3, 4]. This fact implies great difficulty for clinical diagnosis. In addition it is not easy to identify mothers who consumed alcohol during pregnancy because they usually deny it [5, 6]. There are biological markers of chronic consumption such as ethyl glucuronide (Etg) [7, 8] and the ethyl esters of fatty acids (FAEE) [9, 10], which would allow (with laboratory analysis) the identification and early intervention of affected children. The main importance of those chronic consumption markers lies in their wide window of detection; i.e. they remain in the biological samples for much longer than the original compound (in this case, ethanol), giving more scope for the analysis and the detection of consumption. Those biological markers would also allow identifying the low-to-moderate consumption of alcohol during pregnancy, thereby detecting a group of neonates that could be at risk of poor neurodevelopment [1, 11, 12]. At the present time, there is no single laboratory marker capable of demonstrating consumption of alcohol with certainty. Many clinical trials show that an early identification of children exposed to alcohol and/or drug abuse during pregnancy diminishes the disabilities of those children [13–15]. Xenobiotics consumed by women during pregnancy can be identified in the meconium and hair of the newborn [9, 16]. Because of its volatility and its potential absorption from external sources, it is not possible to determine ethanol in hair. Meconium is a fundamental matrix, as it represents a deposit of all agents that the baby has been exposed to during the last trimester of gestation, because it forms after the 12th week [17, 18]. However, it must be taken into account that meconium is a very complex matrix, containing very low concentrations of analytes, and requires previous purification of the sample and sensitive analytic techniques. The objective of this work is to know the prevalence of the positive biomarkers of alcohol in newborns with
prenatal alcohol exposure in our area (covers urban and rural area), as well as establishing the analytic procedures to identify the Etg and the FAEEs in the newborn’s meconium as a routine practice, this practice is not standardized in this moment.
2. Method 2.1. Study population We performed a prospective study during a 10 month period (February–December 2010) in neonates born in the maternity ward of the Hospital Cl´ınico Universitario of Santiago de Compostela. This hospital is a tertiary referral care center for a population of 394,172 people (including rural and urban areas), and has a level 3b Neonatal Unit. During this period 2,594 babies were born. Inclusion criteria were: neonates ≥32 weeks of gestation and/or ≥1500 g weight at birth and belonging to our health area. Exclusion criteria were: babies affected from major malformations or severe diseases, or consent refusal from the parents. Inclusion criteria were met by 2,466 patients. Three babies per day were selected at random, one day each week, during the morning shift. Of these 129 newborns, 19 parents refused consent; thus, finally, 110 babies were included in the study. 2.2. Maternal data The Hospital’s Ethics Committee approved the study, and mothers were asked for informed consent. Study staff gave a survey to the mothers, and the responses were seen by the study staff during the collection process. Questionnaire data was obtained about: mothers’ age, level of education (primary, secondary, university), employment status (stable, unemployed, housewife), drug consumption (alcohol and/or other drugs), over-the-counter medications or mouthwash which contain alcohol, perinatal and obstetric antecedents (gestational controls, previous abortions). Alcohol consumption was evaluated by the system of standard drinking units [19], which sums the number of glasses of wine (∼10 g), bottles of beer (∼10 g) and units of spirits (∼10 g) consumed regularly per week. We consider occasional consumption of alcohol to be 1–2 drinks per week containing 10 g of pure alcohol content each. Habitual consumption would be above this quantity.
A. Ba˜na et al. / Prenatal alcohol exposure
2.3. Newborn data Several data were evaluated for each newborn, such as: gestational age, Apgar score, anthropometric parameters (length, weight, head circumference), neuromuscular evaluation (Ballard’s test). Samples of the first urine and meconium from each newborn were also collected. 2.4. Perinatal levels of drug exposure Urine samples of newborns were analyzed by a quick drug test (screening done by immunoassay) that could alert us of recent drug consumption, such as: cocaine, methadone, opiates, benzodiazepines, cannabis and amphetamine compounds. Positive cases were confirmed by gas chromatography/mass spectrometry (GC/MS). The detection of FAEEs and Etg in meconium was performed by gas chromatography / mass spectrometry (GC/MS) and by liquid chromatography/tandem mass spectrometry (LC/MS/MS) because of the sensitivity and specificity of those techniques, since meconium is a biological sample where the concentration of analytes is very low. The amount of meconium used was 500 mg for each analysis. An analytic method with LC/MS/MS was developed to determinate the presence of Etg in meconium by a linear calibration line at range 50–200 ng/g, where the LOD was 20 ng/g, and the LOQ was 50 ng/g. A deuterated internal standard (ethyl glucuronide d5 ) was used for quantification. After a sample pretreatment with Acetonitrile, we used solid phase (Bond Elut Certify columns) for the extraction procedure. The development of the analytic methodology needed for the determination of FAEEs in meconium, was performed using GC/MS. As extraction technique, we opted for MicrowaveAssisted Extraction (MAE), because it was the most efficient extractive technique and the one that obtained the cleanest extracts. MAE has scarcely been used to extract drugs of abuse from different materials such as serum, urine, tablets and coca leaves. Several authors have shown the extraction capacity and advantages of the MAE [20–23]. The analytical method for Etg and FAEEs was completely validated regarding linearity, limits of detection and quantification, repeatability (intraday precision), reproducibility (interday precision) and recovery, following the recommendations of the FDA [24].
49
Both markers (FAEEs and Etg) were determined when there was sufficient amount of the biological sample available, since it had to be previously divided for the two determinations (this was done, as mentioned above, with different techniques of extraction and analysis). When this was not possible, only one of the markers was chosen for the analysis, depending on the availability of the equipments. The results were considered positive if the FAEE’s value was >1000 ng/g meconium, and if the Etg’s value was >50 ng/g meconium. In the literature there are no clearly defined criteria about the concentration that these markers need to reach in meconium to be considered as positive [8, 25, 26]. In the literature, FAEE concentration of 2 nmol/g (the equivalent to 600 ng/g) is the value established as cut-off for prenatal exposure to alcohol, to ensure that there has been alcohol consumption during pregnancy [8, 25]. We chose a value of 1000 ng/g to avoid false positive results due to endogen FAEEs, as it is known that ethyl myristate is positive in 80% of meconium samples of non-exposed babies [27]. Regarding ethyl glucuronide, there are also different criteria [28, 29]. In our study, such as in Tarcomnicu’s [30], we considered a positive value starting at a concentration of 50 ng/g. The analytical technique used for this immunologic screening of urine to detect drugs of abuse was a COBAS INTEGRA 400® 2.5. Data analysis A descriptive analysis was performed using the SPSS 15.0 programme for Windows. Data are expressed as mean ± SD or as absolute frequencies and percentiles. A linear regression analysis was performed to correlate alcohol consumption and birth weight. Values of p < 0.05 were considered statistically significant.
3. Results One hundred and one valid meconium samples were obtained from the newborns included in the study. Both Etg and FAEEs were determined in 47 cases; whereas, FAEEs or Etg alone, were determined in 35 and 19 cases, respectively. The decision of determining one or both biochemical markers was made according to the amount of sample available. The result was positive for one or two of the markers in 35 cases, which means a frequency of 34.65% of the babies studied; in
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A. Ba˜na et al. / Prenatal alcohol exposure Table 1 Etg and FAEE values on meconium in patients with at least one positive result
Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Ethyl-glucuronide (ng/g meconium)
Fatty acids ethyl esters (ng/g meconium)
No 56 No (–) 86 No 189 No No 63 55 74 218 No (–) No (–) No No No No (–) (–) (–) (–) 73 (–) (–) 95 263 (–) (–) (–) (–) No
5500.30 144.35 1352.30 17462 546.86 17994 7079.70 5377.40 1925 296.54 169.15 1299.10 4311.20 1417 4260.60 1399.50 8969.10 3286.50 1600.80 112427 3039.50 1225.40 99116 7006.50 3543 No 1492.30 2181.30 44470 No 1004.10 2715.60 1881.70 27892 45533
No: not detected; Negative: (–).
8 cases (17%) both markers were positive (Table 1). An overview of the cumulative FAEEs concentrations and Etg concentrations is given in Table 2. Urine quick drug test was performed in all the cases. Three of the urine samples gave a positive result for cocaine, and all three were confirmed by GC/MS. Two of these cases were also positive for alcohol (n 1, 4) without evidence of abuse of other drugs. These positive results were later confirmed with the meconium samples. The method used to obtain this confirmation is the same used in a study published by some of the authors of the present study [31]. None of the newborns included in the study showed remarkable neurological disorders, and the Ballard’s test was concordant to their morphological age.
Table 2 Cumulative fatty acid ethyl esters (FAEEs) and ethyl glucuronide (Etg) concentrations in the 110 meconium samples analyzed Cumulative concentration (FAEEs) (ng/g) (LOD = 100 ng/g) (LOQ = 1000 ng/g) 5001 Etg concentration (ng/g) (LOD = 20 ng/g) (LOQ = 50 ng/g) 50
Number of samples 4 48 18 12
5 13 10
Regarding anthropometric parameters, there were no significant differences between the group of babies exposed to alcohol and the non-exposed; however, a correlation between alcohol consumption and low birth weight was observed, since 20% of the patients that showed a positive result presented low birth weight. This correlation is even greater if we take into account those patients with values of meconium FAEEs over 10000 ng/g meconium (3, 4) (Table 3). However, on a regression analysis after adjusting for potential confounders such as gestational age (p < 0.001), mothers’ smoking habits (p = 0.274) and sex of the newborn (p = 0.070), no statistically significant correlation between markers of alcohol consumption and birth weight was found. Twenty three of the 110 patients studied required admittance to the Neonatal Unit; of which 8 were admitted to the Neonatal Intensive Care Unit. The main causes were: prematurity of 32–35 weeks (7), respiratory distress (6), children of insulin-dependent diabetic mothers (3), congenital malformations (3), and other causes (4). There was no significant association between alcohol exposure and need of admittance to the Neonatal Unit. The 3 patients admitted with a positive result for cocaine showed clinical data that justified the toxicological analysis. One of them was the son of a mother with self-admitted consumption of cocaine during gestation; another one presented intense jitteriness from birth, and the third one was a polimalformative syndrome, associated with cocaine abuse. None of them developed neonatal withdrawal. Regarding the mothers, 91.42% of the mothers with a baby showing a positive result (above the cut-off for Etg or for cumulative FAEEs) denied any consumption and only 5 (4.5%) admitted occasional alcohol consumption during pregnancy. No one admitted frequent
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51
Table 3 Fatty acid ethyl esters (FAEEs) and birthweigth Weight AGA SGA
10000 ng/g meconium
67 13
13 1
4 1
4 3
Spearman’s r = 0.15 (p = 0.182). AGA: weight is appropriate for gestational age; SGA: weight is small for gestational age. Table 4 Maternal features as markers of alcohol screening in children Maternal features
Children with positive alcohol markers (n. 35)
Children with negative alcohol markers (n. 75)
Statistical significance
Age Level of education
31.90 ± 4.4 PE: 14 (40%) SE: 13(37%) UE: 8 (23%) W: 22(63%) U: 6(17%) H: 7(20%) 3 (8.6%) 0 35 (100%) 7 (20%)
32.54 ± 3.7 PE: 16 (21%) SE: 48 (64%) UE: 11(15%) W: 57 (76%) U: 12 (16%) H: 6 (8%) 2 (2.6%) 1 74 (98.6%) 16 (21.3%)
P = 0.89 P = 0.83
Work status
Admission of alcohol’s consumption Admission of illicit drug consumption Controlled Pregnancy Previous abortions
P = 0.7
NA NA P = 0.25 P = 0.382
PE: primary education; SE: secondary education; UE: university education; W: working; U: unemployed; H: housewife. NA: Not applicable.
consumption. None claimed exposure to other sources of alcohol such as over-the-counter medications or mouthwash containing alcohol. Currently, the alcohol content of these external sources is insignificant; thus, the possibility that such minor metabolites of alcohol as Etg or FAEEs reach the meconium is not taken into account. There was no significant association between alcohol consumption and mother’s age (average age of 31.9 ± 4.4 for mothers whose babies showed positive alcohol markers, versus 32.54 ± 3.7 for mothers whose babies showed negative alcohol markers. In mothers under 30 years of age, the markers were positive 24.32% of cases). There was no significant association either between alcohol consumption and level of education or employment status of the mothers. All of the pregnancies were supervised by the midwife/obstetrician (Table 4).
4. Discussion We noticed a high percentage of pregnant women (34.6%) who ingested alcohol during pregnancy as it was reflected by the early detection markers analyzed in their children and confirms other literature finding
that many women do not admit to substance use during pregnancy, as reason that the biomarker approach is valuable. Garc´ıa-Algar [32] already alerted in 2008 about the high prevalence of fetal alcohol exposure (45%) in their study carried out in a low socioeconomic status cohort of a Mediterranean city (Barcelona). Another assay performed in several cities of Italy, shows a considerable variability in the prevalence of fetal exposure to ethanol, reaching a figure of 29.4% in cities like Rome [25]. These data, together with data that we have obtained, indicate that gestational ethanol exposure is widespread, at least in parts of Southern Europe. In a survey carried out among Italian and Spanish neonatologists regarding the awareness of detection of FAS and FASD during pregnancy, approximately 50% of the Italian neonatologists and 40% of the Spanish neonatologists allowed women to drink an occasional glass of wine or beer during pregnancy [33]; it should be emphasaized that small amount of alcohol is dangerous and can lead to neurodevelopmental deficits. In addition to these high percentages, we must add the fact that mothers don’t usually admit to consumption of alcohol or other drugs due to social stigmas associated with such consumption (91.9% of the mothers with a positive result for alcohol consumption in
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A. Ba˜na et al. / Prenatal alcohol exposure
our cohort didn’t admit any kind of toxic abuse during pregnancy). Anyway we do not used a validated test and several screening tools have been validated for use in prenatal care settings, and the most recently proposed brief and easy to use T-ACER3 has demonstrated high sensitivity and specificity in both identifying risk drinking during pregnancy [34]. These last two findings would justify there are not many references regarding the neurodevelopment repercussion of a moderate prenatal exposure to alcohol, FASD (which in younger populations of the USA and some countries of Western Europe might reach to 2–5%) [4]. All this would show the importance of developing methods be a valid supplement to oral screening instruments for analyzing biological markers and allow the early detection of those children at risk of deficient neurodevelopment. This knowledge would permit an early intervention, which would lead to improvement of executive function and emotional problem-solving skills [11, 35]. In our study we determined ethyl glucuronide and/or ethyl esters of fatty acids. These markers, specifically FAEES are validated biomarkers of fetal alcohol exposure [6] but do not permit an accurate quantitative assessment (the quantification criteria are not standardized and they depend on each author’s opinion and of the FAEEs chosen for analysis) but they allow establishing a correlation with alcohol consumption as well as differentiating between occasional consumption and chronic or abusive consumption [7, 8, 16]. Other markers could be of interest, such as phosphathydilethanol [36] and carbohydrate-deficient transferring (CDT) [37]. The determination of phosphathydilethanol in blood is useful because of its wide window of detection (1–3 weeks of abstinence) and it is a feasible procedure [38]. The determination of carbohydrate-deficient transferring (CDT) is useful because it is a surrogate indirect marker that increases in blood when there is chronic alcohol consumption. A worldwide change in drinking habits is being confirmed, and there is a higher consumption among groups within the higher cultural and economic levels, as well as an increase in consumption in women between the ages of 16–24 [39]. In our study, as a consequence of the general tendency to delay the first pregnancy (mean maternal age was 31.4), we didn’t observe a greater consumption among younger mothers. Instead, we did observe an increase in consumption among housewives and university-educated women. Those results are in disagreement with the Pichini
study, where low maternal education was associated with biomarker scores over the cutoff [25]. We observed, as in other studies [40, 41], a correlation between alcohol exposure and low birth weight, present in up to 42.8% of those who exceed a value of FAEE of 5000 ng/g in meconium. The neurological exams didn’t detect assessable abnormalities in the immediate perinatal period, but a further follow up is needed set up strategies for functional assessment, individualized planning, structured teaching, and developments in cognitive-behavioral methods of interventions for those identified to detect some cognitive impairments that could be treated early and thus improve those patients’ evolution as far as possible. We consider that further studies are needed to asses this potential long-term repercussion. Regarding the consumption of others drugs of abuse, analytical determinations in meconium showed that three mothers had consumed cocaine, and two of them had also consumed alcohol. This data agree with the EMCDDA annual report of 2011, which points to Spain as the country with the highest cocaine consumption. Prenatal exposure to cocaine can produce neurotoxic effects and malformations as a result of blood flow interruption and due to its teratogenic action [42, 43]. Two of our three patients with fetal exposure to cocaine showed these alterations probably caused by such exposure. Observed findings indicate that fetal exposure to alcohol has such a high detrimental impact, that it would be worthwhile to make all the necessary efforts to establish routine early diagnosis of the problem.
5. Conclusions Small or moderate alcohol consumption during pregnancy is underestimated. Thus, there is a group of children at risk of suffering neurodevelopment disorders. These children could be benefited by early stimulation and follow up. Ethyl glucuronide and ethyl esters of fatty acids are useful biomarkers that allow detection of alcohol consumption during pregnancy; thus, their use could be standardized. We still cannot determine with certainty which is the best marker, so we propose the use of both, and even of some others such as CDT or phosphatydilethanol, because the presence of endogenous FAEEs may lead to false positive cases, and Etg’s levels are sometimes
A. Ba˜na et al. / Prenatal alcohol exposure
so low that they cannot be detected. As a consequence, we agree with other authors regarding the importance of the analysis of several alcohol consumption markers, because the results obtained using various markers are more reliable.
[14]
[15]
[16]
Financial disclosure The authors have nothing to disclose. [17]
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