Limitations of Nail and Hair Ethyl Glucuronide (Etg) Levels to Assess Maternal Alcohol Use

Nicole AHayes¹, Aileen Baldwin¹, Raquel Magri¹, Erika Ostrander¹, Matthew Smith²and Michael Fleming^1*

¹Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine, Illinois, USA

²School of Social Work, University of Michigan, USA

Received Date: 20 June 2019

Accepted Date:  22 July 2019

Published Date: 29 July 2019

Citation

Hayes NA, Baldwin A, Magri R, Ostrander E, Smith M, et al. (2019) Limitations of Nail and Hair Ethyl Glucuronide (Etg) Levels to Assess Maternal Alcohol Use. Drug Depend Addict 2019(1): 19-25.

Correspondence should be addressed to
Michael Fleming, USA
E-mail: m-fleming@northwestern.edu
DOI: 10.33513/DDAD/1901-03

Copyright

Copyright © 2019 Michael Fleming et al. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and work is properly cited.

Abstract

Alcohol use during pregnancy remains a major public health problem throughout the world. Rates of alcohol use among pregnant women vary from 10-50%. The most important sequelae of maternal alcohol use is the development of fetal alcohol spectrum disorder. Recent population-based surveys suggest rates of 1-5%. The aim of this report is to present the results of a study designed to assess Ethyl Glucuronide (EtG) levels in nail and hair samples collected at the time of delivery in a sample of 253 post-partum women compared to Phosphatidylethanol (PEth) levels. EtG is a direct metabolite of alcohol and can detect alcohol use in the previous 90 days (third trimester of pregnancy in this sample). PEth is formed in the cellular membrane of red blood cells, only in the presence of alcohol and can detect alcohol use in the previous 28 days. The study was conducted at a maternity hospital in Montevideo, Uruguay between 2016-2017. The average age and parity of the women was 28.1 and 2.3 respectively. Of the 253 postpartum women recruited for the study, 18% reported first trimester alcohol use and 8% reported second and third trimester use. EtG was positive (>8pg/mg) in 2.4% of the maternal nail samples and in 0.4% of hair samples. In contrast, positive maternal PEth levels (>8ng/ml) occurred in 44% of the post-partum women. This report suggests EtG levels are of limited value to assess maternal alcohol use during the third trimester or to determine newborn risk for FASD. This study also suggests that EtG levels <7 pg/mg do not confirm abstinence in pregnant women.

Introduction

Guidelines from the US and international countries encourage women to abstain from alcohol consumption during their pregnancies in order to prevent the myriad of negative health consequences for their children[1-3]. Despite these recommendations, many women continue to drink alcohol while pregnant[4]. Some infants born to these women develop Fetal Alcohol Syndrome (FAS) or Fetal Alcohol Spectrum Disorder (FASD), a cluster of symptoms constituting growth delays, dysmorphological facial features, and neuropsychological deficits[5]. Globally, some countries have rates of up to 50% of women drinking during pregnancy, constituting a worldwide health concern[6]. While findings on specific risk factors for alcohol consumption during pregnancy are mixed, age, education level, and socioeconomic status have all been implicated as potential risk factors for increased alcohol use[7-9]. Identification of risk factors and overall use of alcohol during pregnancy is challenging, as historically confirmation of maternal alcohol use during pregnancy relied on self-reported disclosure, often many years post-exposure. These self-reports can be inaccurate due to difficulties with recall as well as the stigma associated with reporting alcohol consumption during pregnancy. In the past few years, researchers have begun to utilize biomarkers to objectively measure maternal alcohol consumption.

Ethyl Glucuronide (EtG) is a direct metabolite of ethanol that is growing in popularity for detecting alcohol use in various populations[10]. EtG is an identifiable biomarker for alcohol in samples of hair, nails, blood, urine, and meconium[11-13]. EtG detection in nail and hair has advantages over other samples due to the longer time frame of detection; EtG is detectable in urine for several days, and is detectable in nail and hair for a period of several months[14,15]. Levels of identified EtG in a sample can differentiate between those who have abstained from alcohol during the identifiable period (concentration less than 7pg/mg), those who have repeated moderate consumption of alcohol during this period (concentration greater than or equal to 7pg/mg), and those who have repeated excessive alcohol consumption (concentration greater than or equal to 30pg/mg)[16]. Studies of alcohol users have demonstrated much higher sensitivity and specificity in high consuming populations[17]. EtG has not been demonstrated to be vulnerable to environmental exposure such as hand sanitizer, meaning that positive results can be assumed to be from oral consumption of alcohol[18]. However, several factors in addition to alcohol consumption can impact a positive EtG result in hair samples. Studies have found hair coloring, bleaching, and shampooing all decrease both the sensitivity and specificity of detection[19]. Research has also found that different methods of extraction of EtG from samples yield different concentrations[11,15]. The clinical utility of EtG to detect maternal alcohol use during pregnancy remains unclear.

EtG has been used previously in studies of prenatal alcohol exposure. Researchers in New Mexico collected hair samples from women midway through their pregnancies to compare objective EtG results to subjective self-reported alcohol use[11]. This study found that EtG had good specificity (86.1%) and it reached 19.1% sensitivity; however, reported alcohol use was lower than expected in the weeks before pregnancy[11]. Studies have begun to compare EtG results from hair and nails in mothers with EtG results in infant meconium samples to confirm the infant exposure to alcohol. However, maternal EtG has not emerged as consistent in the ability to confirm alcohol exposure at the same rates as EtG in infant meconium[20].

Phosphatidylethanol (PEth) is a direct biomarker of alcohol metabolism that has also been shown to be a highly sensitive and specific indicator of alcohol use and exposure and has generated significant interest as a potential objective measurement of prenatal alcohol exposure. PEth is a unique phospholipid formed from phosphatidylcholine present in the membranes of red blood cells, and occurs only in the presence of ethanol through a metabolic process catalyzed by phospholipase D[21-23]. USDTL developed and validated the extraction and detection of PEth from dried blood spots as a screening method for prenatal alcohol exposure[24]. A number of studies in recent years have demonstrated the validity of screening for PEth in dried blood spots for the detection of both maternal alcohol consumption during pregnancy and prenatal alcohol exposure[25-28].

Due to the limited available research on utility of EtG as a confirmatory biomarker in pregnant women, the current study aims to help fill that gap by examining EtG concentrations in maternal nail and hair samples in a cohort of South American women shortly after delivery. These biomarker samples will be compared to women’s self-reported alcohol use and PEth from finger stick blood spot samples to help determine if EtG is a reliable biomarker for detecting alcohol use during pregnancy. Results from this study will help inform clinical practice by potentially adding an objective measure of prenatal alcohol exposure.

Methods

Procedures

Data were obtained from a larger study examining biomarkers of alcohol in pregnant women in Uruguay. Women were approached and consented to the study as they were admitted to the hospital to give birth. A researcher conducted a brief interview of the woman’s health behaviors including alcohol, tobacco, marijuana and cocaine use during pregnancy. The woman then provided a blood spot, and nail and hair sample for biomarker analysis. Samples were collected within 48 hours of delivery. All samples and interview data were de-identified prior to analysis. The study was approved by the Northwestern University and Uruguay Institutional Review Boards (IRB). Women were consented in Spanish. Consent records are stored in the office of Dr. Michael Fleming at Northwestern University. All samples and interviews were exported to the United States for analysis at the United States Drug Testing Laboratories, Inc (USDTL).

Measures

Interview: Age, racial/ethnic identification, marital and employment status, size of their household, prenatal care, gravidity and parity information were obtained from the interview. Women also provided information about their alcohol consumption during each trimester and use three months prior to pregnancy; including frequency, amount, and type of alcohol consumed. In an attempt to minimize underreporting the alcohol use questions were imbedded in questions on diet, tobacco and overall health.

Biosamples: Concentration of nail EtG was obtained from nail clippings with the goal to obtain a minimum of 20 mgs which is equivalent to 2-3 mm with nail thickness the width of a quarter. Hair samples were obtained by cutting a pencil width size clump of hair from the crown, as close to the scalp was possible. The goal was to obtain 200 strands of hair or 100 mgs. The minimum amount of nail and hair needed to test for EtG is 10 mg with 20mg providing enough for confirmatory analyses[24]. For PEth testing, five drops of blood were obtained by finger puncture and placed on a blood spot filter paper card that contained five circles within 24 hours of delivery. Isopropyl alcohol was used to clean the skin before the finger puncture and does not impact the level of PEth detected in blood spot cards. EtG and PEth are stable at room temperature for up to 6 months[28,29].

Hair and nail samples were analyzed for EtG at United States Drug Testing Laboratories using a previously published method[24]. Weighed portions (10-50 mg) of hair (1.5 inches closest to scalp) and fingernail clippings are powdered in a Mini-Bead Beater 8 ball mill using 2.1mm stainless steel beads in 2 mL polypropylene vials. The EtG is extracted from the powdered specimens by adding 1mL DI water to the vials and incubating in a 40°Csonicating water bath and 50 µLEtG_-d5(20 ng/mL) is added as the internal standard. After centrifugation, the decanted extracts are subjected to Solid Phase Extraction (SPE) using a quaternary amine with chloride counter ion SPE cartridge with a 200- mg bed in a 10 mL reservoir (CUQAX12A,United Chemical Technologies, Bristol, PA, USA). The extracts are loaded onto the SPE columns after the columns are conditioned with methanol (2 mL) and DI water and rinsed with 2 mL DI water and 2 mL methanol. The ETG is eluted from the SPE cartridges with 2 mL of 2% formic acid in methanol following a rinse step of 2 mL of DI water and 2 mL of methanol. The eluates are evaporated under a stream of nitrogen at 40°Cand reconstituted in 0.1 mL of DI water. Separation is achieved with a Synergi Polar RP (50 mm × 2.0 mm, 2.5 µm particle size) C-18 column (Phenomenex, Torrence, CA, USA) held at 30°C. The detector is an Agilent 6460 Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) using electro-spray in the negative mode. The internal standard (ETG_-d5) is monitored using the m/z 226.1 > 74.9 (quantification) transition and the m/z 226.1 > 85.2 (qualifying ion) transition.The m/z 221.1 > 74.9 (quantification ion) and m/z 221.1 > 85.0 (qualifying ion) transitions are used to monitor ETG.The limit of detection is 2 pg/mg, the limit of quantitation is 8 pg/mg, and the assay is linear up to 2000pg/mg.

Blood spot cards were analyzed for PEth using a previously published method [24]. Three standard blood spot punches (3.1mm) are prepared for each dried human blood spot specimen, calibrator, and control. The punches are extracted with methanol (1 mL), evaporated under a stream of nitrogen, and the residues are reconstituted in 0.5 mL of mobile phase A (20% 2 mM ammonium acetate: 58% acetonitrile: 22% isopropanol). Separation is achieved with an Agilent Zorbax Eclipse Plus (50 mm x 2.1 mm, 1.8 mm particle size) C-8 column held at 30°Cusing an Agilent 6460 Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) as the detector. The method monitors a single isomer of PEth (palmitoyl/oleoyl), which is the most prevalent PEth species.The limit of detection is 2 ng/mL, the limit of quantitation is 8 ng/mL, and the assay is linear up to 800 ng/mL.

Participants

Data were collected from women who admitted to the Labor and Delivery Clinic at the Military Hospital in Montevideo, Uruguay.All women above the age of 18 were invited to participate in the study.Women were excluded from the study if their infants were born with serious life-threatening birth defects. Women were not compensated for their participation due to hospital regulations.The study was conducted seven days a week and all women admitted to the hospital for delivery were approached by the research staff and invited to participate.All interviews were conducted in Spanish.The research staff who conducted the interviews and collected the biomarker samples were trained by Dr. Fleming and his team how to conduct the interview and collect the samples.All the research staff had a clinical background.Dr. Raquel Magri was the site-PI who is a pediatrician in Uruguay with extensive research and clinical experience.

Data analysis

Interview, biomarker results and medical record data was hand entered by research staff at Northwestern University into a redcap data base. Analyses was performed using SPSS software.The primary analyses were descriptive. Differences between women with positive and negative biomarker results were compared using t-tests. Sensitivity and specificity of EtG compared to PEth or maternal self-report was not determined as the number of positive EtG specimens was too low for a meaningful determination.

Results

The average age of enrolled women was 28.10 years (SD=6.83, Range = 18 to 44 years). 43.0% of the sample were married. The majority of the sample identified as ethnically White (57.37%), with smaller proportions identifying as Mixed race (33.86%), Black Descent (6.37%), and Native Uruguayan (1.99%). The majority of women reported received an incomplete secondary level of education (56.97%). Completed university was uncommon, with only 10.75% having achieved this level of education. 60.15% of the women were employed either full time or part time at the point of their enrollment in the study, and 18.72% of women identified themselves as full-time homemakers. The mean number of pregnancies was 2.32 (Standard deviation=1.54) with the mean number of living children as 1.99 (Standard deviation=1.22). Full demographics are available in table 1.