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. Author manuscript; available in PMC: 2016 Mar 1.
Published in final edited form as: Clin Chem. 2015 Jan 16;61(3):523–532. doi: 10.1373/clinchem.2014.233718

Clinical Sensitivity and Specificity of Meconium Fatty Acid Ethyl Esters, Ethyl Glucuronide, and Ethyl Sulfate for Detecting Maternal Drinking During Pregnancy

Sarah K Himes a, Kimberly A Dukes b, Tara Tripp b, Julie M Petersen b, Cheri Raffo b, Larry Burd a, Hein Odendaal d, Amy J Elliott e, Dale Hereld f, Caroline Signore g, Marian Willinger g, Marilyn A Huestis a; the Prenatal Alcohol in SIDS and Stillbirth (PASS) Networkc
PMCID: PMC4485427  NIHMSID: NIHMS699757  PMID: 25595440

Abstract

Background

We investigated agreement between self-reported prenatal alcohol exposure (PAE) and objective meconium alcohol markers to determine the optimal meconium marker and threshold for identifying PAE.

Methods

Meconium fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) were quantified by liquid chromatography-tandem mass spectrometry in 0.1 g meconium from infants of Safe Passage Study participants. Detailed PAE information was collected from women with a validated timeline follow-back interview. As meconium formation begins during weeks 12-20, maternal self-reported drinking at or beyond 19 weeks was our exposure variable.

Results

Of 107 women, 33 reported no alcohol consumption in pregnancy, 16 stopped drinking by week 19, and 58 drank beyond 19 weeks (including 45 3rd trimester drinkers). There was moderate-substantial agreement between self-reported PAE ≥19 weeks and meconium EtG ≥30 ng/g (kappa: 0.57, 95% CI 0.41-0.73). This biomarker and associated cutoff was superior to a 7 FAEE sum ≥2 nmol/g and all other individual and combination marker cutoffs. With meconium EtG ≥30 ng/g as the gold-standard condition and maternal self-report ≥19 weeks gestation as the test condition, 82% sensitivity (95% CI: 71.6-92.0) and 75% specificity (95% CI: 63.2-86.8) were observed. A significant dose-concentration relationship between self-reported drinks per drinking day and meconium EtG ≥30 ng/g also was observed (P<0.01).

Conclusions

We assessed meconium EtG, EtS, and FAEE concentrations in the same meconium sample and compared concentrations to detailed self-reported PAE data. Maternal alcohol consumption ≥19 weeks was better represented by meconium EtG ≥30 ng/g compared to current FAEE cutoffs.

Keywords: meconium, ethyl glucuronide, fatty acid ethyl ester, pregnancy, alcohol, ethyl sulfate

Introduction

Prenatal alcohol exposure (PAE) is associated with neurodevelopmental, cognitive, and behavioral disabilities in exposed infants (1). Fetal alcohol spectrum disorders (FASD) encompass a continuum of negative consequences including growth retardation, craniofacial dysmorphology and cognitive impairments associated with PAE. Cognitive and behavioral impairments also are prevalent in alcohol-exposed children lacking craniofacial dysmorphology (2). Estimated PAE prevalence in the US is 8% (3), compared to 57% among US Northern Plains American Indians (4) and 34-51% among mixed ancestry women from South Africa's (SA) Western Cape (5). Alcohol consumption during pregnancy is often underreported, challenging PAE identification. PAE biomarkers from delivery (6) would allow for early, proactive intervention for at-risk infants and prevent or mitigate PAE-associated outcomes (1, 7).

Biomarkers in meconium, the 1st neonatal feces, can potentially identify PAE (6, 8-18). Fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) are established PAE markers (6, 8-18); although these meconium markers identify maternal alcohol consumption, analyte cutoff validation and interpretation criteria are limited.

FAEE are formed from endogenous free fatty acids and ethanol by specific and nonspecific enzymes in blood and several tissues (19). FAEE do not cross the placenta; therefore, meconium FAEE result exclusively from fetal synthesis (20). Some recommend summing 7 meconium FAEE (ethyl linolenate, palmitoleate, arachidonate, linoleate, palmitate, oleate, and stearate) and employing a ≥2 nmol/g cutoff for heavy PAE (9, 10), while others sum fewer FAEE (11, 21). Summations often eliminate individual FAEE variability; although, one report demonstrated ethyl oleate and ethyl linoleate meconium concentrations alone distinguished women drinking ≥7 drinks per drinking day (DPDD) from those drinking fewer (8).

EtG is produced by UDP-glucuronosyltransferase-catalyzed conjugation of ethanol and glucuronic acid, while EtS results from ethanol and activated sulfate conjugation by sulfotransferases (22). Unlike FAEE, meconium EtG is primarily of maternal origin as EtG readily crosses the placenta and fetal glucuronidation capacity is limited (23, 24). While relatively little is known about placental EtS transfer, evidence of variable yet significant fetal sulfotransferase activity (25) suggests meconium EtS may be of fetal origin.

There are many published meconium FAEE reports, but far fewer for EtG and EtS. Previously proposed meconium EtG cutoffs were suggested based on meconium FAEE results; meconium FAEE limitations and biases influenced suggested cutoffs, including 111 (16), 200 (16), 274 (11), 333 (14), and 444 (12) ng/g (0.5-2 nmol/g). Meconium EtG and EtS quantification may be superior to FAEE due to improved stability in meconium (26) and insensitivity to maternal diet variation. Our method validation indicated most FAEE in authentic meconium showed degradation after 12 h at room temperature and 72 h at 4°C, with large between-subject variability observed (26). Additionally, freeze/thaw stability experiments showed EtG and EtS concentrations ≤11% of initial results (13, 17, 26). These results suggest FAEE may not be adequate long term meconium alcohol markers due to degradation susceptibility arising from environmental conditions. Additionally, Chan et al. reported that olive oil consumption during pregnancy was associated with increased total meconium FAEE (9).

Clearly more research is needed to validate meconium EtG and EtS cutoffs against reliable self-report measures. Our objective was to evaluate agreement between self-reported PAE and meconium markers. With meconium results as the objective gold-standard condition, we determined sensitivity and specificity performance characteristics with self-reported PAE.

Material and Methods

Participants

The Safe Passage Study of the Prenatal Alcohol in Sudden Infant Death Syndrome (SIDS) and Stillbirth (PASS) Network is a prospective study of 12,000 mother-infant dyads, enrolled during pregnancy and followed 1 year after birth. The primary objective is to determine the relationship between PAE, stillbirth, and SIDS (27). Women were recruited in the US Northern Plains, and Cape Town, SA, representing three diverse populations: American Indians, mixed ancestry, and white (4, 5, 27). 108 meconium samples were selected for this alcohol marker investigation based on maternal self-report in the following categories: 1) no alcohol consumption any time during pregnancy (n=33), 2) average 3rd trimester (>24 weeks) DPDD >10 (n=14), 3) average 3rd trimester DPDD 3-10 (n=32), 4) average 3rd trimester DPDD 0-<3 (n=10), or 5) any drinking during the 1st or 2nd trimesters with no drinking during the 3rd trimester (n=19). All meconium samples were collected within 48 h of birth (some at participants’ homes) and refrigerated as soon as possible. Once obtained by the study sites, samples were immediately frozen (≤ -20°C) and remained frozen until analysis. Samples were transported on dry ice.

Maternal Self-Report

Self-reported PAE was determined by the Timeline Follow-Back (TLFB) method (28), a structured, calendar-based interview collecting detailed ethanol consumption data for each drinking event with respect to alcohol type, container size, sharing and duration (29). The interview was modified for PASS (27) and administered at recruitment (between 6 weeks gestation through delivery), each prenatal visit (20-24, 28-32, and 34-38 weeks), and 1 month post-delivery. At recruitment, women were interviewed about alcohol consumption the year prior to pregnancy and around conception (15 days ± last menstrual period). At other visits, the reporting period was the 30 days prior to the last drinking day (LDD), if the participant consumed alcohol since her last visit. Alcohol brand/type and volume were collected to accurately calculate total grams of alcohol consumed. Number of standard DPDD was calculated from total grams of alcohol consumed each drinking day, based on the US standard drink definition of 14 g ethanol (29). Local standard clinical practices determined gestational age (GA) at each site. Exposure timing was determined from GA at time of maternal report. Meconium formation begins between 12 and 20 weeks (30). Prior studies generally evaluated meconium alcohol biomarkers using maternal self-report from ≥0 weeks. We applied a ≥19 weeks cutoff to account for potential GA measurement variability. Additional measures were created using self-reported PAE at ≥12, ≥20, and ≥28 weeks (an updated definition of 3rd trimester onset implemented after this sample selection). All measures included self-reported drinking categorization and DPDD.

Meconium Analysis

We quantified 9 FAEE (ethyl laurate, myristate, linolenate, palmitoleate, arachidonate, linoleate, palmitate, oleate, and stearate), EtG, and EtS were quantified from a single 0.1 g meconium sample and analyzed by liquid chromatography tandem mass spectrometry method (26). Limits of quantification (LOQs) were 25-50 ng/g for FAEE and 5 and 2.5 ng/g for EtG and EtS, respectively. Sample preparation involved methanolic homogenization with wooden applicator sticks and solid phase extraction (26). Meconium was thoroughly mixed before sampling to ensure a representative sample. Previously, triplicate sampling from three positive sources across three test conditions demonstrated intra-subject variability 0.7-17.6% (26).

Statistical Analysis

We investigated distributions of continuous meconium biomarkers and exposure variables were investigated with Kolmogorov-Smirnov tests and visual boxplot inspection. Bivariate associations between self-reported exposure and meconium biomarkers were assessed using scatterplots, t tests, χ2 chi-square tests, and Spearman correlations; among meconium biomarkers, Spearman correlations were used. Agreement between maternal self-report and meconium biomarkers was evaluated using kappa statistics and corresponding 95% confidence intervals (CI). With meconium markers as the gold-standard condition, clinical sensitivity and specificity performance characteristics and corresponding 95% CI were calculated using self-reported drinking ≥19 weeks as the test. Sensitivity was defined as the number of women reporting drinking ≥19 weeks whose infants’ meconium marker concentrations were ≥cutoff, divided by all infants with meconium marker concentrations ≥cutoff (true positives/true positives and false negatives). Specificity was defined as the number of women who did not report drinking ≥19 weeks and whose infants’ meconium marker concentrations were <cutoff, divided by all infants with meconium marker concentrations <cutoff (true negatives/true negatives and false positives). To evaluate a dose response effect, four variables were created for average DPDD after 19 weeks: 0, >0 to ≤3, >3 to 10, and >10. Multiple logistic regression analysis was performed associating the meconium marker with the best sensitivity and specificity performance with the four variables representing increasing self-reported DPDD (with no DPDD as the reference group), adjusting for GA at LDD or non-drinking day. Model discrimination and calibration were assessed using the c-statistic and Hosmer-Lemeshow test, respectively. All analyses used a two-sided test for statistical significance with P<0.05, except where discussed. Analyses were performed with SAS/STAT software, version 9.3. All analyses used a 2-sided test for statistical significance with P < 0.05, except where discussed.

Results

Participants

Of the 108 women selected, 107 women had maternal self-reported PAE status defined at ≥19 weeks gestation. Thirty-three women reported no alcohol consumption during pregnancy, 16 drank early in pregnancy with cessation by week 19, and 58 continued drinking beyond week 19, with 45 drinking in their 3rd trimester (24% of women reported drinking within 1 month of delivery). Women drinking after 19 weeks reported drinking an average (SD) of 5.6 ± 5.3 and 4.7 ± 4.7 DPDD in the 2nd and 3rd trimesters, respectively.

Meconium Alcohol Marker Prevalence and Concentrations

More samples were positive for EtG (65.4%) compared to EtS (21.5%), each of the 9 FAEE alone (10.3-52.3%), and the three FAEE summations at our LOQs (Table 1). The proposed (9, 10) meconium 7 FAEE summation (excluding ethyl laurate and myristate) cutoff ≥2 nmol/g identified 25 (23.4%) infants, while the 4 FAEE summation (ethyl myristate, palmitate, oleate, and stearate), currently used in hair testing (31), identified 37 (34.6%).

Table 1.

Meconium assay limits of quantification (LOQ) and positive meconium samples’ median and range alcohol marker concentrations

Assay LOQ ng/g (nmol/g) N (%) Median ng/g (nmol/g) Range ng/g (nmol/g)
EtG 5 (0.023) 70 (65.4%) 208 (0.935) 6.2 – 103,716 (0.028 – 467)
EtS 2.5 (0.020) 23 (21.5%) 11 (0.087) 2.8 – 408 (0.022 – 3.23)
Ethyl Laurate 50 (0.219) 11 (10.3%) 81 (0.355) 51 – 884 (0.223 – 3.87)
Ethyl Myristate 25 (0.098) 19 (17.8%) 88 (0.343) 25 – 4,985 (0.098 – 19.4)
Ethyl Linolenate 25 (0.049) 11 (10.3%) 118 (0.385) 25 – 768 (0.082 – 2.51)
Ethyl Palmitoleate 25 (0.053) 17 (15.9%) 354 (1.250) 25 – 9,075 (0.089 – 32.1)
Ethyl Arachidonate 25 (0.045) 32 (29.9%) 73 (0.220) 25 – 3,511 (0.075 – 10.6)
Ethyl Linoleate 25 (0.049) 42 (39.3%) 156 (0.504) 26 – 9,800 (0.084 – 31.8)
Ethyl Palmitate 50 (0.176) 32 (29.9%) 139 (0.487) 50 – 25,080 (0.176 – 88.2)
Ethyl Oleate 25 (0.048) 56 (52.3%) 280 (0.902) 25 – 50,500 (0.081 – 163)
Ethyl Stearate 50 (0.160) 30 (28.0%) 165 (0.528) 52 – 6,688 (0.166 – 21.4)
Sum all 9 FAEE 58 (54.2%) 501 (1.636) 25 – 84,064 (0.081 – 279)
Sum 7 FAEE (excluding laurate and myristate) 58 (54.2%) 501 (1.636) 25 – 78,195 (0.081 – 256)a
Sum 4 FAEE (myristate, palmitate, oleate, and stearate) 58 (54.2%) 375 (1.219) 25 – 61,194b (0.81 – 202)
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a

25 cases had a sum of these 7 FAEE ≥ 2 nmol/g, the suggested cutoff by Chan et al (9, 10).

b

37 cases had a sum of these 4 FAEE ≥ 200 ng/g, a suggested cutoff commonly used in hair FAEE testing (31).

Eighteen samples were both EtS- and EtG-positive; EtG concentrations (19-103,716 ng/g) were 3.3-2,151 times greater than EtS concentrations (2.8-408 ng/g), with a median 285.3 EtG/EtS ratio. There were 5 EtS-positive, EtG-negative samples. In 4 of the 5 cases, EtS was the only detectable marker and concentrations ranged from 3.2-13 ng/g. In the fifth case, 61 ng/g EtS was detected with 13.9 nmol/g summed 7 FAEE.

Spearman correlations between individual and summed meconium alcohol marker concentrations showed EtG was not associated with meconium EtS concentrations and only weakly/negligibly (ρ<0.5, (32)) associated with individual and summed FAEE concentrations (Table 2). An exceptionally strong correlation (ρ≥0.9) was seen between FAEE summations and ethyl linoleate and ethyl oleate, indicating these analytes dominated summation calculations. Most other individual FAEE associations between each other were strong-moderate (ρ=0.5-0.899, (32)). Despite the many moderate/weak correlations, nearly all correlations (85.7%) were significant (n=14, P < 0.00357), on the basis of a Bonferroni-corrected family-wise alpha level (Table 2).

Table 2.

Spearman correlation coefficients (ρ) for meconium marker correlations to each other and summation calculationsa

EtG EtS Summed
EtG EtS		 Ethyl
Laurate		 Ethyl
Myristate		 Ethyl
Linolenate		 Ethyl
Palmitoleate		 Ethyl
Arachidonate		 Ethyl
Linoleate		 Ethyl
Palmitate		 Ethyl
Oleate		 Ethyl
Stearate		 Summed 9
FAEE		 Summed 7
FAEE
EtS 0.2852 (P=0.0029)
Summed EtG EtS 0.9786 (P<0.0001) 0.3830 (P<0.0001)
Ethyl Laurate 0.0999 (P=0.3061) 0.3586 (P=0.0001) 0.1468 (P=0.1314)
Ethyl Myristate 0.2448 (P=0.0110) 0.2559 (P=0.0078) 0.2728 (P=0.0045) 0.6442 (P<0.0001)
Ethyl Linolenate 0.1634 (P=0.0927) 0.3632 (P=0.0001) 0.2124 (P=0.0281) 0.8971 (P<0.0001) 0.7323 (P<0.0001)
Ethyl Palmitoleate 0.2196 (P=0.0230) 0.2365 (P=0.0142) 0.2091 (P=0.0307) 0.5033 (P<0.0001) 0.7530 (P<0.0001) 0.5080 (P<0.0001)
Ethyl Arachidonate 0.3259 (P=0.0006) 0.3751 (P=0.0007) 0.3441 (P=0.0003) 0.4360 (P<0.0001) 0.5926 (P<0.0001) 0.5201 (P<0.0001) 0.5799 (P<0.0001)
Ethyl Linoleate 0.3237 (P=0.0007) 0.2539 (P=0.0083) 0.3400 (P=0.0003) 0.5467 (P<0.0001) 0.7292 (P<0.0001) 0.5622 (P<0.0001) 0.7082 (P<0.0001) 0.7860 (P<0.0001)
Ethyl Palmitate 0.3619 (P=0.0001) 0.3185 (P=0.0008) 0.3760 (P=0.0001) 0.4206 (P<0.0001) 0.7034 (P<0.0001) 0.4469 (P<0.0001) 0.7362 (P<0.0001) 0.7580 (P<0.0001) 0.7979 (P<0.0001)
Ethyl Oleate 0.3738 (P<0.0001) 0.2665 (P=0.0055) 0.3827 (P<0.0001) 0.4953 (P<0.0001) 0.6722 (P<0.0001) 0.5046 (P<0.0001) 0.6727 (P<0.0001) 0.7732 (P<0.0001) 0.9279 (P<0.0001) 0.8018 (P<0.0001)
Ethyl Stearate 0.4882 (P<0.0001) 0.2846 (P=0.0030) 0.4783 (P<0.0001) 0.3049 (P=0.0014) 0.6057 (P<0.0001) 0.3881 (P<0.0001) 0.6381 (P<0.0001) 0.7093 (P<0.0001) 0.6432 (P<0.0001) 0.7809 (P<0.0001) 0.6768 (P<0.0001)
Summed 9 FAEE 0.4217 (P<0.0001) 0.2766 (P=0.0039) 0.4329 (P<0.0001) 0.4908 (P<0.0001) 0.6748 (P<0.0001) 0.5024 (P<0.0001) 0.6630 (P<0.0001) 0.7771 (P<0.0001) 0.9117 (P<0.0001) 0.8158 (P<0.0001) 0.9828 (P<0.0001) 0.7390 (P<0.0001)
Summed 7 FAEE 0.4213 (P<0.0001) 0.2766 (P=0.0039) 0.4325 (P<0.0001) 0.4888 (P<0.0001) 0.6725 (P<0.0001) 0.5004 (P<0.0001) 0.6629 (P<0.0001) 0.7790 (P<0.0001) 0.9114 (P<0.0001) 0.8169 (P<0.0001) 0.9829 (P<0.0001) 0.7398 (P<0.0001) 0.9999 (P<0.0001)
Summed 4 FAEE 0.4299 (P<0.0001) 0.2750 (P=0.0041) 0.4382 (P<0.0001) 0.4733 (P<0.0001) 0.6702 (P<0.0001) 0.4871 (P<0.0001) 0.6684 (P<0.0001) 0.7758 (P<0.0001) 0.9050 (P<0.0001) 0.8244 (P<0.0001) 0.9801 (P<0.0001) 0.7542 (P<0.0001) 0.9989 (P<0.0001) 0.9990 (P<0.0001)
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a

For marker combinations, individual analyte nmol/g concentrations were summed. Summed 7 FAEE, included all except ethyl laurate and myristate, as recommended in meconium (9, 10), and summed 4 FAEE included ethyl myristate, palmitate, oleate, and stearate, as recommended in hair (31).

b Strong (ρ=0.7-0.899) and exceptionally strong correlations (ρ=0.9-1.0) are shown in bold (32).

Agreement Between Self-Report and Meconium Markers

The meconium marker with the highest agreement with self-reported PAE ≥19 weeks was EtG ≥30 ng/g, with a kappa value of 0.57 (95% CI: 0.41-0.73), signifying moderate to substantial agreement (Table 3). Individual FAEE at their LOQs had kappa values ≤0.24, indicating only slight agreement. In comparison, both FAEE combinations of all 9 and 7 (excluding ethyl laurate and myristate) at ≥2 nmol/g demonstrated lower agreement with self-reported PAE than individual FAEE markers or the summation of 4 FAEE (ethyl myristate, palmitate, oleate, and stearate) ≥200 ng/g. Agreement was higher between self-reported PAE ≥19 weeks and marker combinations that included EtG. Among the 58 women reporting drinking after 19 weeks, 48 were EtG-positive with a median (range) concentration of 1,101 ng/g (7.2-103,716), 16 were EtS-positive with a median concentration of 14 ng/g (2.8-408), and 37 were positive for one or more FAEE with 7 FAEE summed concentrations ranging from 0.081-256 nmol/g (only 13 were ≥2 nmol/g).

Table 3.

Clinical sensitivity and specificity of maternal self-reported drinking during pregnancy at or beyond 19 weeks gestation (test condition) compared to individual and combined meconium alcohol markers (gold-standard condition).

Meconium analytes and cutoffs Sensitivity (95% CI) Specificity (95% CI) Kappa (95% CI)
Ethyl Laurate ≥ 50 ng/g (LOQ) 54.5 (25.1, 84.0) 45.8 (35.9, 55.8) 0.001 (−0.106, 0.109)
Ethyl Myristate ≥ 25 ng/g (LOQ) 57.9 (35.7, 80.1) 46.6 (36.2, 57.0) 0.025 (−0.111, 0.161)
Ethyl Linolenate ≥ 25 ng/g (LOQ) 54.5 (25.1, 84.0) 45.8 (35.9, 55.8) 0.001 (−0.106, 0.109)
Ethyl Palmitoleate ≥ 50 ng/g (LOQ) 52.9 (29.2, 76.7) 45.6 (35.3, 55.8) −0.008 (−0.138, 0.123)
Ethyl Arachidonate ≥ 25 ng/g (LOQ) 62.5 (45.7, 79.3) 49.3 (38.0, 60.6) 0.096 (−0.069, 0.261)
Ethyl Linoleate ≥ 25 ng/g (LOQ) 61.9 (47.2, 76.6) 50.8 (38.6, 62.9) 0.119 (−0.060, 0.298)
Ethyl Palmitate ≥ 25 ng/g (LOQ) 71.9 (56.3, 87.5) 53.3 (42.0, 64.6) 0.204 (0.043, 0.366)
Ethyl Oleate ≥ 25 ng/g (LOQ) 62.5 (49.8, 75.2) 54.9 (41.2, 68.6) 0.174 (−0.012, 0.361)
Ethyl Stearate ≥ 50 ng/g (LOQ) 76.7 (61.5, 91.8) 54.5 (43.4, 65.7) 0.243 (0.087, 0.399)
Sum 9 FAEE ≥ 2 nmol/g 53.8 (34.7, 73.0) 45.7 (34.8, 56.5) −0.003 (−0.159, 0.152)
Sum 7 FAEE ≥ 2 nmol/g (9, 10) 52.0 (32.4, 71.6) 45.1 (34.4, 55.9) −0.02 (−0.173, 0.134)
Sum 4 FAEE ≥ 200 ng/g (31) 64.9 (49.5, 80.2) 51.4 (39.7, 63.1) 0.144 (−0.028, 0.316)
EtS ≥ 2.5 ng/g (LOQ) 69.6 (50.8, 88.4) 50.0 (39.3, 60.7) 0.126 (−0.018, 0.271)
EtG ≥ 5 ng/g (LOQ) 68.6 (57.7, 79.4) 73.0 (58.7, 87.3) 0.386 (0.214, 0.558)
EtG ≥ 10 ng/g 72.3 (61.4, 83.2) 73.8 (60.5, 87.1) 0.448 (0.279, 0.617)
EtG ≥ 15 ng/g 73.4 (62.6, 84.3) 74.4 (61.4, 87.5) 0.468 (0.300, 0.636)
EtG ≥ 25 ng/g 80.4 (70.0, 90.8) 74.5 (62.5, 86.5) 0.550 (0.391, 0.708)
EtG ≥ 30 ng/g 81.8 (71.6, 92.0) 75.0 (63.2, 86.8) 0.569 (0.413, 0.725)
EtG ≥ 50 ng/g 82.4 (71.9, 92.8) 71.4 (59.6, 83.3) 0.535 (0.376, 0.693)
EtG ≥ 333 ng/g (1.5 nmol/g) (14) 96.8 (90.6, 100.0) 63.2 (52.3, 74.0) 0.476 (0.334, 0.619)
EtG ≥ 444 ng/g (2 nmol/g) (15, 18) 96.7 (90.2, 100.0) 62.3 (51.5, 73.2) 0.459 (0.317, 0.601)
EtG ≥ 5 ng/g, or EtS ≥ 2.5 ng/g 66.7 (56.0, 77.3) 75.0 (60.0, 90.0) 0.362 (0.192, 0.531)
EtG ≥ 10 ng/g, or EtS ≥ 2.5 ng/g 70.0 (59.3, 80.7) 75.7 (61.9, 89.5) 0.424 (0.255, 0.593)
EtG ≥ 15 ng/g, or EtS ≥ 2.5 ng/g 71.0 (60.3, 81.7) 76.3 (62.8, 89.8) 0.444 (0.276, 0.612)
EtG ≥ 30 ng/g, or EtS ≥ 2.5 ng/g 74.6 (63.9, 85.4) 75.0 (62.2, 87.8) 0.488 (0.322, 0.654)
EtG ≥ 50 ng/g, or EtS ≥ 2.5 ng/g 74.6 (63.5, 85.7) 70.8 (58.0, 83.7) 0.453 (0.284, 0.623)
EtG ≥ 333 ng/g, or EtS ≥ 2.5 ng/g 83.3 (72.1, 94.6) 64.6 (53.0, 76.2) 0.449 (0.289, 0.610)
Sum 9 FAEE ≥ 2 nmol/g, or EtG ≥ 1.5 nmol/g 74.5 (62.0, 86.9) 61.7 (49.4, 74.0) 0.352 (0.180, 0.525)
Sum 9 FAEE ≥ 2 nmol/g, or EtG ≥ 1.5 nmol/g, or EtS ≥ 2.5 ng/g 70.4 (58.2, 82.5) 62.3 (49.2, 75.3) 0.327 (0.148, 0.505)
Sum 7 FAEE ≥ 2 nmol/g, or EtG ≥ 1.5 nmol/g 74.5 (62.0, 86.9) 61.7 (49.4, 74.0) 0.352 (0.180, 0.525)
Sum 7 FAEE ≥ 2 nmol/g, or EtG ≥ 1.5 nmol/g, or EtS ≥ 2.5 ng/g 70.4 (58.2, 82.5) 62.3 (49.2, 75.3) 0.327 (0.148, 0.505)
Sum 4 FAEE ≥ 200 ng/g, or EtG ≥ 1.5 nmol/g 74.5 (62.5, 86.5) 64.3 (51.7, 76.8) 0.386 (0.213, 0.559)
Sum 4 FAEE ≥ 200 ng/g, or EtG ≥ 1.5 nmol/g, or EtS ≥ 2.5 ng/g 70.7 (59.0, 82.4) 65.3 (52.0, 78.6) 0.36 (0.183, 0.537)
Sum 9 FAEE ≥ 2 nmol/g, or EtG ≥ 30 ng/g 74.2 (63.3, 85.1) 73.3 (60.4, 86.3) 0.47 (0.302, 0.637)
Sum 9 FAEE ≥ 2 nmol/g, or EtG ≥ 30 ng/g, or EtS ≥ 2.5 ng/g 70.6 (59.8, 81.4) 74.4 (60.7, 88.1) 0.426 (0.256, 0.596)
Sum 7 FAEE ≥ 2 nmol/g, or EtG ≥ 30 ng/g 74.2 (63.3, 85.1) 73.3 (60.4, 86.3) 0.47 (0.302, 0.637)
Sum 7 FAEE ≥ 2 nmol/g, or EtG ≥ 30 ng/g, or EtS ≥ 2.5 ng/g 70.6 (59.8, 81.4) 74.4 (60.7, 88.1) 0.426 (0.256, 0.596)
Sum 4 FAEE ≥ 200 ng/g, or EtG ≥ 30 ng/g 75.0 (64.4, 85.6) 76.7 (64.1, 89.4) 0.506 (0.342, 0.670)
Sum 4 FAEE ≥ 200 ng/g, or EtG ≥ 30 ng/g, or EtS ≥ 2.5 ng/g 71.4 (60.8, 82.0) 78.4 (65.1, 91.6) 0.463 (0.297, 0.628)
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CI indicates confidence interval

Performance Characteristics of Self-Reported Exposure and Meconium Markers

With EtG ≥30 ng/g as the gold-standard condition, self-reported PAE ≥19 weeks clinical sensitivity and specificity were 81.8% (95% CI: 71.8-92.0) and 75.2% (95% CI: 63.2-86.8), respectively. Self-reported PAE ≥19 weeks yielded similar performance with EtG and EtS at their LOQs for sensitivity (68.6%, 95% CI: 57.7-79.4; 69.6%, 95% CI: 50.8-88.4, respectively), although when EtG was ≥5 ng/g, self-reported PAE ≥19 weeks had higher specificity (73.0%, 95% CI: 58.7-87.3) compared to EtS ≥2.5 ng/g (50.0%, 95% CI: 39.3-60.7). Self-reported PAE ≥19 weeks demonstrated 64.9% specificity (95% CI: 49.5-80.2) and 51.4% specificity (95% CI: 39.7-63.1) with the 4 FAEE summation (ethyl myristate, palmitate, oleate, and stearate) ≥200 ng/g; these performance measures were higher than those for the 7 and 9 FAEE summations.

With meconium EtG ≥30 ng/g as the gold-standard condition, there were 45 true positives (positive self-report, EtG-positive meconium), 39 true negatives (negative self-report, EtG-negative meconium), 13 false positives (positive self-report, EtG-negative meconium), and 10 false negatives (negative self-report, EtG-positive meconium) (Table 4). Among false positive samples (n=13), women reported LDD between 19.3 and 37.4 weeks. Three had detectable EtG (7.2-24 ng/g) below the ≥30 ng/g cutoff (1 had some individual FAEE detected), 2 others were EtS-positive only, 2 others had some individual FAEE detected, and another had a 7 FAEE sum >2 nmol/g (10.6 nmol/g); no markers were detected in the other 5 (Table 4). Of the 10 self-report-positive, EtG-negative samples, only 5 women reported 3rd trimester drinking; on LDD median GA was 33.6 (30.6-37.4) weeks and DPDD was 7.0 (0.8-13.8) standard drinks. Among the 5 women reporting 2nd but no 3rd trimester drinking, on LDD median GA was 25.9 (19.3-27.1) weeks and DPDD 1.6 (0.1-11.6) standard drinks, respectively. All 10 women reported 1-3 drinking days during the 31 days prior to LDD. Six of 10 samples had summed 7 FAEE meconium concentrations ≥2 nmol/g (Table 4).

Table 4.

Meconium alcohol marker results and maternal self-reported alcohol consumption for true and false positives and negatives considering meconium EtG ≥30 ng/g as the gold-standard condition and maternal self-reported alcohol consumption beyond 19 weeks as the test condition.

Meconium EtG (gold-standard condition) Self-reported alcohol intake week 19-delivery (test condition) n Median (range) meconium EtG (ng/g) Additional meconium marker results Median (range) gestational age (GA) at last reported drinking daya (weeks) Median (range) standard drinks at last reported drinking day Median (range) number of drinking days in past 31 days from last reported drinking day
True Positives ≥ 30 ng/g + 45 1,215 (30 – 103,716) 14 (31.1%) also EtS-positive; 12 (26.7%) also FAEE-positive with sum 7 > 2 nmol/g; 8 (17.8%) positive for all 3 markers 34.1 (20.1 – 40.9) 3.7 (0.06 – 23.3)b 2 (1 – 31)
True Negatives < 30 ng/g – 39 0 (0 – 26) 9 EtG only; 2 EtS only; 4 FAEE only; 1 EtS and FAEE; 2 EtG and EtS; 1 EtG, EtS, and FAEE 1.1 (−23.7 to 10.3)c 5.6 (1.6 – 15.1)c 2 (1 – 28)c
False Positives < 30 ng/g + 13 0 (0 – 24) 3 of 13 had detectable EtG below cutoff (7.2, 21, 24 ng/g)d; 2 others were EtS-positive only (3.2, 13 ng/g); 1 other FAEE-positive only, with sum 7 FAEE > 2 nmol/g. Given positive maternal self-report, these cases should be classified as exposed. 33.4 (19.3 – 37.4) 2.3 (0.14 – 13.8) 1 (1 – 9)
False Negatives ≥ 30 ng/g – 10 79.5 (51 – 6,230) 5 also with 7 FAEE sum ≥ 2 nmol/g and 1 EtS- and FAEE-positive (6,230 ng/g EtG, 13 ng/g EtS, 4.44 sum 7 FAEE nmol/g).e Strong intrauterine alcohol exposure evidence in 6 of 10 cases as multiple markers were in agreement; women likely underreported drinking behavior. 5.3 (−7.9 to 16.9)f 5.0 (0.5 – 12.0)f 2 (1 – 31)f
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a

GA at birth based on fetal ultrasound (n=73, 68.2%) or last reported menstrual cycle date (n=34, 31.8%) at prenatal enrollment visit; GA at last reported drinking calculated from subtracting last reported drinking day from GA at birth. Negative numbers indicate last drinking day was pre-conception.

b

Data available for 44 (97.8%).

c

Data available for 17 (43.6%). The 22 remaining women reported no drinking in any trimester.

d

The 3 EtG-positive samples were from women who reportedly all stopped drinking during week 34. Women whose infants had the 3.2 and 13 ng/g EtS-positive samples stopped drinking during week 20 and 37, respectively. The woman whose infant had meconium 7 FAEE sum > 2 nmol/g stopped drinking at 27.1 weeks.

e

The false negative with 6,230 ng/g EtG, 13 ng/g EtS, and high FAEE reportedly stopped drinking at 5 weeks gestation. During the 31 days prior to her stop, she drank every day with an average of 7 drinks/day reported.

f

Data from 7 women (70%) were included here. The remaining 3 women reported no drinking in any trimester.

Dose-Concentration Relationships

A statistically significant dose-concentration relationship was observed between self-reported DPDD after 19 weeks and meconium EtG ≥30 ng/g, adjusted for GA at LDD (model P<0.0001, DPDD all P<0.01, GA P=0.42); the model had good discrimination (c-statistic=0.81) and was well calibrated (χ2=9.3, P=0.23). For self-reported DPDD between >0 and ≤3, the odds of observing meconium EtG ≥30 ng/g was 9.1 (95% CI: 1.8-45.8) times higher than that for women report no drinking. Odds of meconium EtG ≥30 ng/g increased respectively to 22.6 (95% CI: 6.2-82.0) and 29.4 (95% CI: 2.9-295.6) when considering self-reported DPDD between >3 and ≤10 and >10 DPDD, compared to women reporting no drinking. Results were similar when maternal self-reported drinking cutoffs were ≥12, ≥20, or ≥28 weeks, largely due to most women reporting both 2nd and 3rd trimester drinking (2nd but not 3rd trimester, n=13; 3rd but not 2nd trimester, n=8). Only one woman was classified differently between the 19 and 20 week cutoffs; dose-concentration results for both cutoffs were similar.

Discussion

Less is known about EtG and EtS meconium markers compared to FAEE, and few studies compared PAE detection capability among markers. Our study is the most thorough comparison to date of meconium EtG and EtS concentrations to FAEE concentrations and detailed, prospective maternal self-report during pregnancy. Five prior studies collected self-reported PAE and meconium EtG, EtS and FAEE. However, high underreporting in 4 did not permit authors to compare self-report with meconium EtG and EtS; instead, meconium EtG and EtS cutoffs were optimized based on meconium FAEE comparison (12, 14, 15, 18). In these studies, the proposed EtG meconium cutoffs (274, 333, and 444 ng/g) were derived from comparisons to summed 5-7 meconium FAEE concentrations (11, 12, 14). This approach is inadequate due to FAEE limitations and bias, including instability and diet concerns. A recent Franconian Maternal Health Evaluation Studies report suggested maternal self-report better agreed with meconium EtG than individual FAEE, supporting FAEE should not be used as the precedent (33). Our study is unique in its simultaneous FAEE, EtG, and EtS quantification in the same 0.1 g meconium sample, achievable due to our combined analytical method (26), permitting accurate clinical sensitivity and specificity comparisons between self-reported exposure and meconium markers.

Our high clinical sensitivity and specificity indicate that accurate PAE classification in infants is optimal with EtG ≥30 ng/g. We found a significant dose-concentration relationship between maternal self-reported average DPDD after 19 weeks and meconium EtG ≥30 ng/g, providing evidence that this marker accurately represents increasing PAE. Our results suggest meconium EtG ≥30 ng/g is the most effective cutoff for PAE identification. DPDD, not timing of consumption, was significantly associated with meconium EtG ≥30 ng/g.

Prior studies demonstrated self-reported exposures could suffer from limited information regarding timing, quantity, and frequency and recall bias in retrospective investigations. The TLFB is a widely accepted methodology to collect detailed and reliable self-reported exposure (29, 34). This method was tailored to our studied populations and modified to collect more precise, serial information throughout pregnancy, allowing evaluation of drinking patterns across time (27). Because of the study design (e.g., women may enter the study anytime during pregnancy) and calendar-based method used to assess self-reported exposure, the number of days where PAE status was defined varied across participants. Therefore, average DPDD in a trimester was selected as the most appropriate measure for this analysis.

Our clinical sensitivity and specificity evaluation indicated maternal self-reported PAE agreed best with meconium EtG ≥30 ng/g (81.8% sensitivity, 75.0% specificity, 0.569 kappa). The currently accepted 7 FAEE summation ≥2 nmol/g cutoff only achieved 52.0% sensitivity and 45.1% specificity. When considering marker cutoff combinations, meconium EtG alone ≥30 ng/g proved superior; of the 13 samples with EtG <30 ng/g from infants of women who reported any drinking beyond 19 weeks only 1 had a 7 FAEE summed concentration ≥2 nmol/g. The better performance of EtG compared to FAEE may have resulted from FAEE limitations including post-collection meconium instability (26) and olive oil variability in maternal diet (9). Weak correlations between EtG and individual and summed FAEE concentrations (Table 2) may reflect the markers’ different formation pathways and possibly different meconium incorporation mechanisms.

Previous meconium EtG and EtS reports did not find EtS alone in meconium; when detected, EtS was always present with EtG (12, 14). However, EtS is often present without EtG in urine (35, 36). We report the first EtS-positive, EtG-negative meconium samples. In one case, 61 ng/g EtS was detected with high FAEE (13.9 nmol/g summed 7 FAEE). In the other 4 cases, EtS was the only detectable marker. This may be explained by our low EtS LOQ compared to other markers, although our EtS LOQ was higher than previously reported (13). EtG-negative meconium also may have resulted from reduced maternal EtG formation due to genetic UGT polymorphisms (37) or liver damage from heavy alcohol consumption. UGT polymorphism studies indicated cannabinoids, specifically cannabidiol, negatively impacted ethanol glucuronidation activity (37). EtS-positive, EtG-negative meconium could result from increased fetal sulfotransferase activity compared to fetal glucuronidation capacity. Fetal liver sulfotransferase activity varies based on isoform and gestational period (25); some isoforms demonstrate higher activity during gestation compared to infancy (25), whereas fetal UDP-glucuronosyltransferase activity is limited, with most activity beginning after birth (23). Urinary EtG/EtS studies demonstrated bacteria-infected urine was capable of both EtG hydrolysis and formation (38); urinary bacterial meconium contamination may explain some positive self-report, EtG-negative meconium (EtG hydrolysis) and negative self-report, EtG-positive meconium (EtG formation). Urine and postmortem tissue EtS was stable (38, 39), providing a possible explanation for some EtS only positive urine. Most SA samples were collected at home within 48 h and returned to the study site; bacterial contamination during home collection could also explain these findings.

Variability in EtG and EtS formation and placental transfer of ethanol and these markers may explain the wide EtG/EtS ratio range (3.3-2151) observed. The low 3.3 EtG/EtS ratio resulted from 19 ng/g EtG and 5.7 ng/g EtS from a self-reported nondrinker. The highest EtG/EtS ratio resulted from 18,286 ng/g EtG and 8.5 ng/g EtS from a PAE infant exposed to 12.7 and 5.6 DPDD in the 2nd and 3rd trimesters, respectively.

Our study had some limitations. Our small feasibility design, versus a true random selection study, and population demographics require the proposed meconium EtG ≥30 ng/g cutoff be validated in larger samples from other populations. A larger sample also would allow investigation of EtS in PAE identification.

Our study demonstrates the importance of identifying PAE via both self-report and objective biomarkers. Meconium biomarkers primarily capture drinking from 3rd and some 2nd trimester exposure, while maternal self-report can identify earlier gestational exposure missed by meconium. In our study, most negative self-report, EtG-positive meconium cases demonstrated strong PAE evidence; EtG's validity was confirmed by additional meconium markers’ presence. These women likely underreported their drinking behavior, demonstrating limitations of self-report reliance, or results could be explained by consumption of other products containing ethanol not identified with our TLFB assessment or bacterial contamination at collection. Given sample size limitations, we cannot appropriately discern whether marker concentrations increasingly represented exposures proximal to birth or whether increasing amounts of EtG-negative meconium diluted EtG-positive meconium, which may explain our observed positive self-report, EtG-negative meconium results.

In conclusion, maternal self-report was correlated with meconium EtG, EtS, and FAEE concentrations in the same 0.1 g meconium sample. Optimal clinical sensitivity (81.8%) and specificity (75.0%) were observed between maternal self-reported alcohol consumption after 19 weeks gestation and meconium EtG ≥30 ng/g. These data should help inform clinicians, clinical chemists and toxicologists on meconium alcohol marker interpretation and PAE identification.

Acknowledgements

The authors gratefully acknowledge cooperation of the study participants, PASS investigators and NICHD Advisory Safety Monitoring Board members: Elizabeth Thom, PhD

(Chair), Reverend Phillip Cato, PhD, James W Collins, Jr, MD, MPH, Terry Dwyer, MD, MPH, George Macones, MD, Philip A May, PhD, Jeff Murray, MD, Richard M Pauli, MD, PhD, Raymond W Redline, MD, and Michael Varner, MD.

List of abbreviations in order cited

PAE

prenatal alcohol exposure

FAEE

fatty acid ethyl esters

EtG

ethyl glucuronide

EtS

ethyl sulfate

FASD

fetal alcohol spectrum disorder

SA

South Africa

DPDD

drinks per drinking day

SIDS

sudden infant death syndrome

PASS

Prenatal Alcohol in SIDS and Stillbirth Network

TLFB

timeline follow-back

LDD

last drinking day

GA

gestational age

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