Newborn Testing For Alcohol Biomarkers

FAEE in Meconium: Back to Square One

by Adam Negrusz, Ph.D., F-ABFT

Alcohol (ethyl alcohol, ethanol) is one of the most commonly used and abused drugs in the world, and currently ranks among the top leading risk factors for global burden of disease, disability and death. According to the multi-state, CDC-sponsored case-control National Birth Defects Prevention Study (NBDPS), approximately 30% of pregnant women reported drinking alcohol during the pregnancy, 8.3% of which reported binge drinking. After the first month of pregnancy, 22.5% of women reported alcohol consumption, 2.7% of women were drinking during all trimesters of pregnancy, and 7.9% during the third trimester.1 The prevalence of a very serious conglomerate of developmental and cognitive disabilities known as Fetal Alcohol Spectrum Disorders (FASD) is estimated to be as high as 2 to 5% of school children, making prenatal alcohol exposure the most preventable source of birth defects in the U.S. Prolonged exposure to high levels of alcohol in utero may result in more serious array of symptoms called Fetal Alcohol Syndrome (FAS). As Dr. Stefan Maxwell reported in the Winter/Spring 2016 issue of NeoTox, The Centers for Disease Control and Prevention (CDC) estimated that FAS occurs at a rate of 0.2-1.5 per 1000 children, while the Institute of Medicine’s estimate is even higher – 0.5-3 newborns per 1000 children.2    

Early diagnosis of prenatal alcohol exposure by testing the specimens collected from the neonate allows for intervention services to be immediately provided to the affected newborn. Meconium is the first stool passed by the neonate usually within the first 48 hours after birth. It begins to accumulate in the fetus’s intestine between 12 and 16 weeks of pregnancy. The excretion of meconium can be significantly delayed in premature, unhealthy, or low birth neonates.3 Some direct biomarkers, ethyl alcohol metabolites, accumulating in meconium have been studied as objective measures of prenatal alcohol exposure. Fatty acid ethyl esters (FAEEs) were the first suggested for that purpose in the early 1990s with the proposed summed cutoffs ranging from 0.5 to 2 nmol/g.3,4 They are nonoxidative metabolites of ethanol formed by an enzymatic esterification of ethyl alcohol with free fatty acids and other lipids by FAEE synthase and acetyl-CoA/ethanol O-acyl-transferase.5 There are more than 20 different compounds in that group,5 but the most frequently mentioned FAEEs are ethyl laurate, myristate, palmitoleate, linolenate, arachidonate, linoleate, palmitate, oleate, and stearate.6 It has been shown that late meconium samples collected from non-exposed neonates contained FAEEs above 2 nmol/g comparing to first-collected specimens that tested negative. The earliest FAEE- positive meconium sample was collected slightly over 18 hours postpartum. The same increase in FAEE content was seen in full-term healthy neonates as well as the neonates from high risk group. The authors conclude that in order to ensure accuracy of results, all efforts should be made to collect the earliest meconium sample.3 In addition, the FAEE false positive results may occur when the samples are collected after the first day of life and/or if several bowel movements already took place. In the same study, incubation of meconium collected from neonates born to nondrinking mothers with glucose or ethyl alcohol resulted in significantly elevated concentrations of FAEEs.3  Similar experiments performed in our laboratory (unpublished data) also revealed significant in vitro formation of FAEEs in meconium samples.

In response to FAEEs limitations, the United States Drug Testing Laboratories, Inc. (USDTL) continued a search of an ‘ideal’ marker for in utero alcohol exposure. In the recent years, ethyl glucuronide (EtG) and ethyl sulfate (EtS), both ethyl alcohol Phase II metabolites, have been studied as viable biomarkers of in utero exposure to alcohol.6, 7, 8, 9 Unlike FAEEs, EtG showed an association with alcohol history.7 In another study, the highest clinical sensitivity and specificity with EtG ≥30 ng/g of maternal self report at ≥ 19 weeks’ gestation was observed comparing to FAEEs and EtS. Only for EtG, a significant dose-meconium concentration correlation was seen (EtG≥30 ng/g).6 None of the studies, however, addressed the issue of in vitro formation of EtG in meconium over proposed cutoff 30 ng/g potentially leading to false interpretation of maternal drinking behavior. In order to answer all relevant questions, our laboratory conducted the experiment on in vitro formation of EtG in meconium samples when exposed to alcohol. Twenty authentic meconium specimens were selected. The analytical method for EtG in meconium consisted of sample homogenization, strong anion exchange solid phase extraction, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The aliquots of ethanol-fortified meconium samples were weighted out and analyzed for EtG at 24- and 48-hour time points. The results clearly show that significant amounts of EtG can be synthesized in meconium when environmentally exposed to alcohol. For that reason it is not an ideal biomarker for maternal alcohol use during pregnancy.10 As part of our research showed, EtS can also be synthesized in meconium samples exposed to external contamination with alcohol, but to the lesser extent. Since a wide range of EtG/EtS ratio in meconium was observed, relying exclusively on EtS presence in meconium may result in a significant number of false negative results.6 In other words, many kids exposed to alcohol in utero could be missed.

In conclusion, since the available markers in meconium for in utero exposure to alcohol have previously mentioned limitations, and based on recent literature data, USDTL decided to tighten meconium collection protocols when FAEE testing is requested. The laboratory will not test the meconium for FAEE if the specimen was collected more than 18 hours after birth. If the baby does not pass the meconium within the first 18 hours postpartum, the sample will be tested for drugs of abuse, but not for FAEE. There are, however, always two other options of testing the neonate for alcohol exposure. The umbilical cord tissue can be tested for EtG, or you can test dried blood spots for phosphatidylethanol (PEth).

References

1. Ethen, M.K., Ramadhani, T.A., Scheuerle, A.E., Canfield, M.A., Wyszynski, D.F., Druschel, C.M., & Romitti, P.A. (2009). Alcohol consumption by women before and during pregnancy. Matern Child Health J, 13(2), 274-285. doi: 10.1007/s10995-008-0328-2

2. Maxwell, S. (2016). Overcoming obstacles in identifying prenatal alcohol exposure. NeoTox, 7(1), 8-11

3. Zelner, I., Hutson, J.R., Kapur, B.M., Feig, D.S., Koren, G. (2012). False-positive meconium test results for fatty acid ethyl esters secondary to delayed sample collection. Alcohol Clin Exp Res, 36(9), 1497-1506. doi: 10.1111/j.1530-0277.2012.01763.x

4. Klein, J., Karaskov, T., & Gideon, K. (1999). Fatty acid ethyl esters: A novel biologic marker for heavy in utero ethanol exposure: A case report. Ther Drug Monit, 21(6), 644-648.

5. Cabarcos, P., Alvarez, I., et al. (2016). Determination of direct alcohol markers: A review. Anal Bioanal Chem, 407(17), 4907-4925. doi: 10.1007/s00216-015-8701-7

6. Himes, S.K., Dukes, K.A., Tripp, T., Petersen, J., Raffo, C., Burd, L., Odendaal, H., Elliott, A.J., Hereld, D., Signore, C., Willinger, M., & Huestis, M.A. (2015). Clinical sensitivity and specificity of meconium fatty acid ethyl ester, ethyl glucuronide, and ethyl sulfate for detecting maternal drinking during the pregnancy. Clin Chem, 61(3), 523-532. doi: 10.1373/clinchem.2014.233718

7. Goecke, T.W., Burger, P., Fasching, P.A., Bakdash, A., Engel, A., Haberle, L., Voigt, F., Faschingbauer, F., Raabe, E., Maass, N., Rothe, M., Beckmann, M.W., Pragst, F., & Kornhuber, J. (2014). Meconium indicators of maternal alcohol abuse during pregnancy and association with patient characteristics. Biomed Res Int, 2014: 702848.

8. Morini, L., Marchei, E., Vagnarelli, F., Garcia-Algar, O., Groppi, A., Mastrobattista, L., & Pichini, S. (2010). Ethyl glucuronide and ethyl sulfate in meconium and hair-potential biomarkers of intrauterine exposure to ethanol. Forensic Sci Int, 196(1-3), 74-77. doi: 10.1016/j.forsciint.2009.12.035

9. Joya, X., Marchei, E., Salat-Batlle, J., Garcia-Algar, O., Calvaresi, V., Pacifici, R., & Pichini, S. (2016). Fetal exposure to ethanol: Relationship between ethyl glucuronide in maternal hair during pregnancy and ethyl glucuronide in neonatal meconium. Clin Chem Lab Med, 54(3), 427-435. doi:10.1515/cclm-2015-0516

10. Shu, I., Baldwin, A., Sagnia, V., Jones, M., Jones, J., Lewis, D., & Negrusz, A. (2016). In vitro formation of ethyl glucuronide in meconium samples. ToxTalk, 40(1), 7-9. doi: 10.1373/clinchem.2014.233718

Dr. Adam Negrusz is the Laboratory Director at USDTL and an Adjunct Associate Professor in the Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago. Negrusz has over 33 years of experience in academic forensic toxicology and drug analysis which has resulted in the publication of nearly 60 articles, few book chapters, nearly 70 abstracts presented at scientific meetings, over 30 professional analytical chemistry reports for sponsors and many standard operating procedures.