Abstract
BACKGROUND
Few investigations have used placenta as an alternative matrix to detect in utero drug exposure, despite its availability at the time of birth and the large amount of sample. Methadone-maintained opioid-dependent pregnant women provide a unique opportunity to examine the placental disposition of methadone and metabolite [2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP)], to explore their correlations with maternal methadone dose and neonatal outcomes, and to test the ability to detect in utero exposure to illicit drugs.
METHODS
We calculated the correlations of placental methadone and EDDP concentrations and their correlations with maternal methadone doses and neonatal outcomes. Cocaine- and opiate-positive placenta results were compared with the results for meconium samples and for urine samples collected throughout gestation.
RESULTS
Positive correlations were found between placental methadone and EDDP concentrations (r = 0.685), and between methadone concentration and methadone dose at delivery (r = 0.542), mean daily dose (r = 0.554), mean third-trimester dose (r = 0.591), and cumulative daily dose (r = 0.639). The EDDP/methadone concentration ratio was negatively correlated with cumulative daily dose (r = 0.541) and positively correlated with peak neonatal abstinence syndrome (NAS) score (r = 0.513). Placental EDDP concentration was negatively correlated with newborn head circumference (r = 0.579). Cocaine and opiate use was detected in far fewer placenta samples than in thrice-weekly urine and meconium samples, a result suggesting a short detection window for placenta.
CONCLUSIONS
Quantitative methadone and EDDP measurement may predict NAS severity. The placenta reflects in utero drug exposure for a shorter time than meconium but may be useful when meconium is unavailable or if documentation of recent exposure is needed.
The placenta exchanges nutrients, oxygen, and waste products; synthesizes hormones, peptides, and steroids; and protects the developing fetus by limiting xenobiotic transfer from the mother. A drug’s physico-chemical properties determine its transfer across the placenta (1 ), with passive diffusion being the main transfer mechanism for lipophilic compounds. Placental metabolism may modify the amounts and types of transferred xenobiotics (2 ), but placental enzymes have low activities compared with those in maternal or fetal liver (1 ). For ethical and safety reasons, few in vivo studies have assessed the transfer and disposition of drugs in human placenta (2 ). Although placental distribution is known from animal models for some drugs, the results are difficult to extrapolate to humans (1, 2 ).
Methadone, the primary opioid-dependence pharmacotherapy used during pregnancy, reduces fetal heroin exposure, obstetric complications, and neonatal morbidity and mortality (3 ). To date, there are no data on the concentrations of methadone and its primary metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP),5 in placenta and whether their concentrations correlate with the methadone dose. Methadone-assisted pharmacotherapy for opioid dependence provides a useful model for investigating drug disposition after in utero drug exposure and the relationship between placental concentrations and neonatal outcomes. The child exposed to a drug in utero may experience neonatal abstinence syndrome (NAS), due to the abrupt end of exposure at birth (4 ). The identification of neonates at risk for more severe NAS could improve infant care.
Our primary objectives were to assess the placental disposition of methadone and EDDP in opioid-dependent pregnant women and to explore the correlations with maternal methadone dose, placental methadone concentration, and neonatal outcome. To better understand the advantages and limitations of drug monitoring in placental tissue, we also compared the results in this unique cohort for methadone, opioid, cocaine, and their main metabolites in placenta with those in matched neonatal meconium samples and with thrice-weekly urine drug test results obtained throughout gestation.
Methods
HUMAN PARTICIPANTS AND NEONATAL OUTCOMES
Participants were recruited from 2 clinical trials (A and B) conducted by the Center for Addiction and Pregnancy, Johns Hopkins Bayview Medical Center (JHBMC) in Baltimore, Maryland. The JHBMC and National Institute on Drug Abuse Institutional Review Boards approved the studies. Participants provided written informed consent. Study A examined the prevalence of mood and anxiety disorders in opioid-dependent pregnant women and the impact of these disorders on methadone treatment success (5), whereas study B compared methadone- and buprenorphine-assisted therapy during pregnancy (6 ). Participants initially received 30 mg methadone daily (Mallinckrodt Chemical); the dosage was increased to 60 mg/day over 4 days. Further dosing adjustments were made as clinically indicated.
Estimated gestational age at birth, birth weight, length, head circumference, and 1- and 5-min Apgar scores were extracted from hospital records. NAS was assessed for the first 10 days of life with a 19-item modified Finnegan scale (6 ). Time to NAS onset was the time in hours from birth until the first score >4, time to NAS peak was the time in hours from birth to peak score, and NAS duration was the time in hours from the first score >4 until all scores were <5.
PLACENTA COLLECTION AND ANALYSIS
Placenta samples were stored at −20 °C until analysis for methadone, EDDP, morphine, codeine, 6-acetylmorphine, cocaine, and benzoylecgonine (BE) by a validated liquid chromatography–ion trap mass spectrometry method (7 ). We initially analyzed 7 placenta samples at 4 different locations (1, 4, 6, and 10 cm from the umbilical cord) to assess whether analyte distribution was homogeneous throughout the matrix and therefore whether analysis of a single intermediate location would accurately represent placental disposition.
MECONIUM COLLECTION AND ANALYSIS
We collected meconium samples from the neonates of mothers providing placental samples until the appearance of milk stool. The samples were frozen until analysis, as for placenta samples, and were analyzed for the same analytes plus m-hydroxybenzoylecgonine (mOHBE), either in house (8, 9 ) or (for opiates, cocaine, and benzodiazepines in some early samples) by the United States Drug Testing Laboratories (10 ).
URINE COLLECTION AND ANALYSIS
To document relapse to opioid and cocaine use, we collected observed thrice-weekly urine samples from the mothers who provided placental samples, from the time of enrollment through birth. Urine samples were analyzed immediately on site with the Abuscreen ONTRAK rapid assays for drugs of abuse (Roche Diagnostic Systems), with cutoffs of 300 μg/L for opiates (morphine) and cocaine (BE) (11 ). Positive opiate and cocaine results were confirmed by gas chromatography–mass spectrometry. Methadone dosing was observed to ensure compliance.
STATISTICAL ANALYSIS
For statistical analyses, we used SPSS for Windows (version 17.0; SPSS). The Kolmogorov–Smirnov test evaluated the fit of data to a normal distribution, and Pearson correlation analysis assessed relationships among placental methadone and EDDP concentrations, EDDP/methadone concentration ratios, maternal methadone dose, and neonatal outcomes (except for 5-min Apgar scores, for which Spearman correlation analysis was applied because of data nonnormality). Pearson correlation analysis was also used to evaluate the relationship between methadone and EDDP concentrations, and EDDP/methadone concentration ratios in placenta and meconium. A statistical probability (P) value <0.05 was considered significant.
Results
HUMAN PARTICIPANTS AND METHADONE DOSING
Placenta samples, demographic data, and methadone-dosing data were available for 19 pregnant opioid-dependent women receiving methadone-assisted therapy. Placenta samples from 3 additional participants were collected and used to test methadone and EDDP distribution across the placenta; however, these samples were not included in further data analysis because maternal methadone-dosing and neonatal-outcome data were not available for these participants. Maternal demographic and methadone-dosing data are shown in Table 1. All pregnancies produced single births, for a total of 19 infants. Most women entered the study during the second trimester between weeks 10 and 29. The mean (SD) estimated gestational age on admission was 20.8 (5.5) weeks.
Table 1.
Demographics and methadone-dosing data for 19 pregnant opioid-dependent women receiving methadone-assisted pharmacotherapy.
| Participant no. |
Age, years |
Race | Years of education |
No. of cigarettes/day |
EGA,a weeks |
Days in study |
Initial dose, mg |
Dose at delivery, mg |
Mean daily dose, mg |
Mean third-trimester daily dose, mg |
Cumulative dose, mg |
Cumulative third-trimester dose, mg |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 19 | AA | 12 | 10 | 17 | 176 | 30 | 60 | 59.5 | 60.0 | 8980 | 4800 |
| 2 | 30 | AA | 11 | 0 | 16 | 176 | 40 | 85 | 74.1 | 81.2 | 12 964 | 8040 |
| 3 | 32 | AA | 14 | 0 | 11 | 200 | 30 | 70 | 66.3 | 70.0 | 13 335 | 6160 |
| 4 | 33 | AA | 12 | 8 | 25 | 104 | 30 | 45 | 44.7 | 45.0 | 4695 | 4230 |
| 5 | 32 | AA | 12 | 20 | 14 | 102 | 30 | 75 | 71.5 | 75.0 | 6010. | 1800 |
| 6 | 34 | AA | 10 | 10 | 23 | 111 | 90 | 110 | 100.4 | 102.5 | 11 245 | 8100 |
| 7 | 31 | AA | 12 | 0 | 10 | 132 | 30 | 90 | 73.6 | 89.7 | 9795 | 3945 |
| 8 | 19 | W | 11 | 20 | 20 | 121 | 30 | 65 | 63.1 | 64.5 | 6565 | 6195 |
| 9 | 25 | B | 12 | 20 | 20 | 141 | 40 | 70 | 81.3 | 86.1 | 11 540 | 7405 |
| 10 | 21 | W | 8 | 40 | 20 | 113 | 40 | 70 | 70.0 | 73.7 | 7980 | 4495 |
| 11 | 34 | AA | 9 | 3 | 24 | 127 | 50 | 80 | 70.5 | 74.7 | 9300 | 7990 |
| 12 | 35 | W | 12 | 0 | 29 | 65 | 60 | 80 | 67.5 | 67.5 | 4590 | 4590 |
| 13 | 32 | AA | 12 | 20 | 25 | 53 | 50 | 30 | 29.6 | 30.0 | 1715 | 1200 |
| 14 | 30 | AA | 12 | 0 | 23 | 124 | 50 | 70 | 65.5 | 68.6 | 8320 | 6650 |
| 15 | 31 | AA | 10 | 8 | 24 | 81 | 50 | 90 | 79.2 | 86.8 | 6815 | 5205 |
| 16 | 23 | O | 10 | 10 | 28 | 76 | 20 | 60 | 37.9 | 37.9 | 3070 | 3070 |
| 17 | 25 | AA | 10 | 14 | 22 | 102 | 40 | 95 | 80.5 | 85.7 | 8450 | 5910 |
| 18 | 26 | W | 13 | 20 | 17 | 139 | 40 | 100 | 86.5 | 97.0 | 12 110 | 6690 |
| 19 | 34 | AA | 9 | 10 | 27 | 89 | 60 | 85 | 69.9 | 69.9 | 3215 | 3215 |
| Mean | 28.7 | — | 11.1 | 11.2 | 20.8 | 117.5 | 42.6 | 75.3 | 68.0 | 71.9 | 7931.3 | 5246.8 |
| SD | 5.3 | — | 1.5 | 10.4 | 5.5 | 38.4 | 15.9 | 19.0 | 16.6 | 19.0 | 3462.7 | 2046.7 |
| Median | 31.0 | — | 12.0 | 10.0 | 22.0 | 113.0 | 40.0 | 75.0 | 70.0 | 73.7 | 8320.0 | 5205.0 |
EGA, estimated gestational age at admission; AA, African American; W, white; B, biracial; O, other.
DISTRIBUTION OF METHADONE AND EDDP ACROSS THE PLACENTA
We measured methadone and EDDP concentrations in 4 placental locations across 7 test placenta samples. Methadone and EDDP were distributed homogeneously across the placenta (see File 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol57/issue3). Concentrations varied <15% for both analytes in 5 placentas (participants 1, 10, 20, 21, and 22); they varied <30% in the other 2 placentas (participants 3 and 9), owing to the greater variation in the concentration of 1 of the analytes at 1 location in 1 sample. Illicit drugs also were evenly distributed in placenta. Cocaine and/or BE were found in all 4 placental locations in 2 participants, whereas morphine and codeine were identified in all sites in 1 placenta. Because of the homogeneous distributions in these 7 test placentas, 1 intermediate location was analyzed for all the other placenta samples.
PLACENTAL METHADONE AND EDDP DISPOSITION AND CORRELATION WITH METHADONE DOSE
Methadone and EDDP concentrations were measurable in all placenta samples after chronic daily methadone dosing (Table 2). The concentrations of methadone were 10-fold to 20-fold greater than for EDDP, with a mean EDDP/methadone concentration ratio of 0.11 (0.06). Statistically significant positive correlations were found for methadone and EDDP concentrations in placenta (r = 0.685; P = 0.001). The placental methadone concentration was significantly correlated with the cumulative daily methadone dose (r = 0.639; P = 0.003). Similar significant correlations also were found between the placental methadone concentration and the methadone dose at delivery (r = 0.542; P = 0.017), the mean daily dose (r = 0.554; P = 0.014), and the mean dose during the third trimester (r = 0.591; P = 0.008). The mean EDDP/methadone concentration ratio was also significantly correlated (but negatively) with the cumulative daily methadone dose (r = 0.541; P = 0.017). Although the correlations were significant, the correlation coefficients themselves ranged from 0.542 to 0.685; the clinical significance of these weak correlations is not yet known.
Table 2.
Methadone (MTD) and EDDP concentrations and EDDP/MTD concentration ratios in 19 placenta and 17 matched meconium samples from 19 opioid-dependent pregnant women receiving methadone-assisted therapy.
| Placenta
|
Meconium
|
|||||
|---|---|---|---|---|---|---|
| Participant no. | MTD, ng/g | EDDP, ng/g | EDDP/MTD | MTD, ng/g | EDDP, ng/g | EDDP/MTD |
| 1 | 1198.6 | 95.9 | 0.09 | 5235 | 80 503 | 15.4 |
|
| ||||||
| 2 | 1909.0 | 142.2 | 0.07 | 5047 | 38 675 | 7.7 |
|
| ||||||
| 3 | 1985.9 | 103.9 | 0.05 | 9864 | 33 352 | 3.4 |
|
| ||||||
| 4 | 1092.4 | 105.6 | 0.10 | 6373 | 44 369 | 7.0 |
|
| ||||||
| 5 | 2647.0 | 517.4 | 0.20 | 2184 | 53 142 | 24.3 |
|
| ||||||
| 6 | 2627.0 | 302.0 | 0.11 | —a | — | — |
|
| ||||||
| 7 | 1784.2 | 78.4 | 0.04 | 2548 | 22 900 | 9.0 |
|
| ||||||
| 8 | 405.2 | 71.7 | 0.18 | 85 | 6375 | 75.0 |
|
| ||||||
| 9 | 1441.6 | 86.9 | 0.06 | 2492 | 13 188 | 5.3 |
|
| ||||||
| 10 | 1372.1 | 105.5 | 0.07 | — | — | — |
|
| ||||||
| 11 | 1724.3 | 213.5 | 0.12 | 18 840 | 62 600 | 3.3 |
|
| ||||||
| 12 | 549.2 | 96.1 | 0.18 | 5481 | 34 753 | 6.3 |
|
| ||||||
| 13 | 308.0 | 35.9 | 0.12 | 2555 | 10 546 | 4.1 |
|
| ||||||
| 14 | 1511.6 | 120.7 | 0.08 | 5785 | 32 826 | 5.7 |
|
| ||||||
| 15 | 1538.2 | 312.3 | 0.20 | 11 334 | 53 800 | 4.7 |
|
| ||||||
| 16 | 1553.9 | 147.8 | 0.10 | 4997 | 28 628 | 5.7 |
|
| ||||||
| 17 | 1048.6 | 90.4 | 0.09 | 5009 | 64 800 | 12.9 |
|
| ||||||
| 18 | 2643.5 | 189.0 | 0.07 | 21 902 | 57 200 | 2.6 |
|
| ||||||
| 19 | 537.3 | 78.6 | 0.15 | 7281 | 57 600 | 7.9 |
|
| ||||||
| Mean | 1467.2 | 152.3 | 0.11 | 6883.1 | 40 897.5 | 11.8 |
|
| ||||||
| SD | 720.4 | 115.4 | 0.05 | 5804.6 | 20 922.8 | 17.2 |
|
| ||||||
| Median | 1511.6 | 105.5 | 0.10 | 5235.0 | 38 675.0 | 6.3 |
Dashes indicate no testing because matched meconium samples were unavailable.
COMPARISON OF METHADONE AND EDDP CONCENTRATIONS IN MATCHED PLACENTA AND MECONIUM SAMPLES
Methadone was the primary analyte in placenta; EDDP concentrations were much lower (Table 2). In meconium, however, EDDP concentrations were 100- to 800-fold greater than in placenta. Methadone concentrations in meconium exceeded those in placenta in 15 of 17 matched samples, and EDDP concentrations always were much higher in meconium than in placenta. Moreover, the EDDP/methadone concentration ratios were reversed, with ratios >0.2 occurring in placenta and much higher ratios (>2.6) in meconium. Neither methadone and EDDP concentrations nor EDDP/ methadone concentration ratios in placentas were correlated with those in matched meconium samples.
NEONATAL OUTCOMES
Neonatal outcomes for the 19 infants born to the 19 opioid-dependent pregnant women receiving methadone-assisted pharmacotherapy are shown in Table 3. The majority of infants (52.6%) were delivered at full term (≥37 weeks), with a mean estimated gestational age at birth of 36.3 (3.4) weeks (range, 30.4 – 41.0 weeks). Three of the 4 infants considered to be of low birth weight (<2500 g) had head circumferences less than typical (<32 cm) for healthy newborns, and all infants with lengths shorter than typical (<45 cm) were preterm. All infants experienced NAS, although only 6 required treatment. Peak NAS scores ranged from 6 –19, the time to NAS onset varied from 3.5 to 127.8 h, and the percentage of NAS scores >4 ranged from 6.3% to 90.1%. Apgar scores at 1 min and 5 min were within reference values (≥7) in all cases.
Table 3.
Neonatal outcome measures for infants (n = 19) of opioid-dependent mothers receiving methadone-assisted therapy.
| Participant no. | EGA,a weeks | Weight, g | Head circumference, cm | Length, cm | Apgar score (1 min) | Apgar score (5 min) | Time to NAS onset, h | Peak NAS score | Time to peak NAS score, h | NAS duration, h | NAS scores >4, % |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 38.3 | 2385 | 30.5 | 43.5 | 8 | 9 | 19.4 | 8 | 61.1 | 61.7 | 69.6 |
| 2 | 41.0 | 3140 | 34 | 51.0 | 8 | 9 | 16.0 | 8 | 16.0 | 25.0 | 66.7 |
| 3 | 38.9 | 4810 | 36 | 52.0 | 9 | 9 | 38.2 | 11 | 60.0 | 22.2 | 22.2 |
| 4 | 40.4 | 4370 | 36 | 51.0 | 8 | 9 | 12.7 | 6 | 30.7 | 45.0 | 28.6 |
| 5 | 30.4 | 1505 | 26 | 41.0 | 9 | 9 | 13.0 | 10 | 13.0 | 96.0 | 31.6 |
| 6 | 38.0 | 2515 | 30 | 48.0 | 9 | 9 | 23.2 | 6 | 23.2 | 48.0 | 13.6 |
| 7 | 33.3 | 2125 | 30 | 43.5 | 8 | 9 | 40.4 | 8 | 40.4 | 12.0 | 25.0 |
| 8 | 40.0 | 4025 | 35.5 | 49.0 | 9 | 9 | 26.0 | 9 | 69.7 | 59.5 | 47.8 |
| 9 | 39.3 | 2865 | 32 | 46.0 | 8 | 8 | 19.0 | 7 | 30.0 | 556.0 | 84.5 |
| 10 | 35.0 | 2740 | 32 | 50.0 | 8 | 8 | 54.0 | 6 | 54.0 | 22.0 | 18.5 |
| 11 | 40.0 | 2675 | 34 | 48.0 | 8 | 9 | 127.8 | 7 | 166.3 | 49.0 | 6.3 |
| 12 | 37.0 | 3110 | 34 | 50.0 | 7 | 9 | 30.0 | 15 | 75.8 | 58.5 | 12.7 |
| 13 | 32.0 | 2080 | 32 | 45.0 | 9 | 9 | 6.3 | 10 | 119.6 | 113.3 | 66.7 |
| 14 | 39.0 | 3760 | 34.5 | 51.0 | 9 | 9 | 23.5 | 11 | 30.5 | 86.7 | 55.8 |
| 15 | 36.0 | 3650 | 33 | 52.5 | 9 | 9 | 28.5 | 19 | 194.0 | 289.5 | 66.7 |
| 16 | 34.0 | 2745 | 33 | 48.0 | 8 | 8 | —b | 4 | 16.5 | — | — |
| 17 | 31.0 | 2850 | 33 | 47.0 | 8 | 9 | 60.7 | 8 | 80.7 | 154.5 | 51.4 |
| 18 | 35.0 | 3180 | 34 | 52.0 | 7 | 9 | 13.3 | 14 | 77.3 | 320.5 | 90.1 |
| 19 | 32.0 | 2995 | 33 | 49.0 | 8 | 9 | 3.5 | 14 | 89.5 | 341.0 | 89.7 |
| Mean | 36.3 | 3027.6 | 32.8 | 48.3 | 8.3 | 8.8 | 30.9 | 9.5 | 65.7 | 131.1 | 47.1 |
| SD | 3.4 | 816.3 | 2.4 | 3.3 | 0.7 | 0.4 | 28.6 | 3.8 | 49.8 | 148.5 | 28.0 |
| Median | 37.0 | 2865.0 | 33.0 | 49.0 | 8.0 | 9.0 | 23.4 | 8.0 | 60.0 | 60.6 | 49.6 |
EGA, estimated gestational age at birth.
Dashes indicate data not available.
PLACENTAL METHADONE AND EDDP CONCENTRATIONS AND CORRELATION WITH NEONATAL OUTCOMES
Placenta EDDP concentration was significantly negatively correlated with newborn head circumference (r = −0.579; P = 0.009), whereas the EDDP/methadone concentration ratio was significantly positively correlated with peak NAS score (r = 0.513; P = 0.030). No other significant correlations were detected. The clinical significance of these correlations is not yet known.
OPIATES AND COCAINE IN MATCHED PLACENTA, MECONIUM, AND THRICE-WEEKLY URINE SAMPLES
Cocaine and opiate results for 19 placenta samples were compared with those for 15 matched meconium samples and 18 sets of thrice-weekly urine results (Table 4). The small amount of meconium collected for 2 samples permitted only methadone and EDDP measurements.
Table 4.
Morphine (MOR), codeine (COD), cocaine (COC), BE, and mOHBE concentrations in matched placenta and meconium samples.
| Urinea
|
|||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Opiates
|
COC
|
Meconium, ng/g
|
Placenta, ng/g
|
||||||||||||
| Participant no. | 2nd TRI, % | 3rd TRI, % | Time from last positive, days | 2nd TRI, % | 3rd TRI, % | Time from last positive, days | MOR | COD | COC | BE | mOHBE | MOR | COD | COC | BE |
| 1 | 17.6 | 6.7 | 34 | 5.9 | 6.7 | 34 | 37.0 | Nb | N | N | 119.0 | N | N | N | N |
|
| |||||||||||||||
| 2 | 3.0 | 0.0 | 176 | 3.0 | 0.0 | 176 | N | N | N | N | 104.0 | N | N | N | N |
|
| |||||||||||||||
| 3 | —c | — | — | — | — | — | N | N | N | N | 68.0 | N | N | N | N |
|
| |||||||||||||||
| 4 | 25.0 | 0.0 | 104 | 25.0 | 0.0 | 104 | N | N | N | N | N | N | N | N | N |
|
| |||||||||||||||
| 5 | 3.0 | 0.0 | 99 | 0.0 | 0.0 | AN | — | — | — | — | — | N | N | N | N |
|
| |||||||||||||||
| 6 | 0.0 | 14.7 | 18 | 0.0 | 0.0 | AN | — | — | — | — | — | N | N | N | N |
|
| |||||||||||||||
| 7 | 2.8 | 0.0 | 128 | 0.0 | 0.0 | AN | 2281.0 | N | N | N | N | N | N | N | N |
|
| |||||||||||||||
| 8 | 0.0 | 0.0 | AN | 0.0 | 0.0 | AN | — | — | — | — | — | N | N | N | N |
|
| |||||||||||||||
| 9 | 94.1 | — | — | 88.2 | — | — | N | N | 233.0 | 969.0 | 254.0 | 39.8 | 2.9 | 7.3 458.9 | |
|
| |||||||||||||||
| 10 | 16.7 | 63.6 | 3 | 0.0 | 0.0 | AN | — | — | — | — | — | N | N | N | N |
|
| |||||||||||||||
| 11 | 55.5 | 0.0 | 118 | 0.0 | 4.4 | 62 | N | N | N | N | 12.2 | N | N | N | N |
|
| |||||||||||||||
| 12 | STT | 10.7 | 61 | STT | 0.0 | AN | 10.0 | N | N | N | 26.0 | N | N | N | N |
|
| |||||||||||||||
| 13 | 100.0 | 0.0 | 42 | 33.3 | 0.0 | 46 | 10.2 | N | N | N | 24.3 | N | N | N | N |
|
| |||||||||||||||
| 14 | 18.2 | 0.0 | 120 | 0.0 | 0.0 | AN | N | N | N | N | N | N | N | N | N |
|
| |||||||||||||||
| 15 | 33.3 | 0.0 | 75 | 0.0 | 0.0 | AN | 14.0 | N | N | N | 12.5 | N | N | N | N |
|
| |||||||||||||||
| 16 | STT | 6.7 | 27 | STT | 63.3 | 25 | 43.0 | N | N | 4.3 | 200.3 | N | N | N | N |
|
| |||||||||||||||
| 17 | 25.0 | 7.1 | 15 | 31.2 | 25.0 | 13 | 2555.0 | 16.3 | 4.4 | 62.0 | 57.3 | N | N | N | N |
|
| |||||||||||||||
| 18 | 18.7 | 0.0 | 100 | 0.0 | 0.0 | AN | N | N | N | N | N | N | N | N | N |
|
| |||||||||||||||
| 19 | STT | 11.5 | 31 | STT | 7.7 | 31 | 65.9 | N | N | 2.4 | 155.1 | N | N | N | N |
Percentages of thrice-weekly matched urine test results positive for opiates and cocaine in the second (2nd TRI) and third (3rd TRI) trimesters, and the time from the last positive urine sample until birth (Time from last positive) are included for comparison of the detection of illicit drug use in the different matrices.
N, negative results (i.e., below the limit of quantification); AN, always negative; STT, participant started in third trimester.
Dashes indicate data not available.
In utero cocaine exposure was confirmed in 11 meconium samples by the presence of cocaine, BE, and/or mOHBE, whereas cocaine exposure was identified in only 1 placenta sample. Placenta and meconium BE concentrations were higher than cocaine concentrations. The range of concentrations for the 11 cocaine-positive meconium samples was 12.2–254.0 ng/g for mOHBE, 2.4 –969.0 ng/g for BE, and 4.4 –233.0 ng/g for cocaine. In the single cocaine-positive placenta sample, the concentrations of BE and cocaine were 458.9 ng/g and 7.3 ng/g, respectively; mOHBE was not included in the placental analytical panel for cocaine and metabolites.
In utero opiate exposure was identified in 8 meconium samples; however, only a single placenta sample was opiate positive (Table 4). In meconium, the range of morphine concentrations was 10 –2555 ng/g; only 1 sample had detectable codeine (16.3 ng/g). Morphine concentrations exceeded codeine concentrations by >10-fold, and no 6-acetylmorphine was present. Morphine and codeine concentrations in the only positive placenta sample were 39.8 ng/g and 2.9 ng/g, respectively, also with no 6-acetylmorphine detectable. Interestingly, no opiates were detected in the meconium of this participant’s newborn.
Thrice-weekly urine data for opiates and cocaine during the second and/or third trimesters of pregnancy were available for 18 of the participants who provided placenta samples. These data confirmed opiate consumption during the second and/or third trimesters of pregnancy for 17 of the participants and confirmed cocaine consumption for 9 participants. Of particular interest is the number of days since the last positive urine test result. These data provide information on the timing of last drug use, which suggests the window for detecting drugs in placenta and meconium. Clearly, many placenta samples were negative for opiates and cocaine while the results of urine and meconium tests were indicating drug relapse. We should note that the fact that mOHBE was not included in the placenta analysis could have contributed to the fewer number of positive placenta samples. The urine data confirmed all of the meconium results, except for the detection of mOHBE in meconium from participants 12 and 15. The lack of positive urine data could have been due to consumption of low cocaine amounts or missed urine samples. Interestingly, for the one participant with a placenta sample positive for opiates, no opiates were detected in the matched meconium sample. Unfortunately, urine data from the third trimester were unavailable for this case.
The remaining 18 placenta samples did not contain measurable opiate or cocaine biomarkers, despite urine screening results indicating drug use by 8 mothers during the third trimester, within 2 weeks before delivery in some participants (the urine results indicated opiate consumption 3 days before delivery by participant 10, and opiate and cocaine consumption 15 and 13 days, respectively, before delivery by participant 17). These data suggest that placenta has a much shorter window of drug detection than meconium.
Discussion
Methadone, EDDP, opiates, and cocaine were homogeneously distributed in the placenta, suggesting that only a single sampling is necessary. High interindividual variation in placental methadone and EDDP concentrations was observed. This finding is not surprising, given the differences in length of study enrollment, gestational age at admission, and cumulative dose throughout pregnancy. Additional factors, including the participants’ age and race, also could have contributed to variations in analyte concentrations. The placental concentrations of methadone (308 –2647 ng/g) and EDDP (35.9 –517.4 ng/g) were higher than commonly observed in maternal plasma (methadone, 45–757.8 μg/L; EDDP, 15– 46.5 μg/L) after ranges of methadone doses (20 –90 mg) similar to those of our study (30 –110 mg) (12–15 ), results that suggest analyte retention. Placental methadone retention was previously noted in vitro by Nekhayeva et al., who assessed methadone transfer across artificially perfused placenta samples (16 ). In addition, methadone and EDDP concentrations in cord blood (arterial–venous blood: 28.7–35.7 μg/L and 8 –9 μg/L for methadone and EDDP, respectively, with 40 mg methadone daily) (12 ) and infant plasma (2.2– 8.1 μg/L and 1.9 μg/L for methadone and EDDP, respectively, with 50 –105 mg methadone daily) (17, 18 ) were lower than in maternal plasma and placenta, indicating a role for the placenta in protecting the fetus from methadone exposure. Furthermore, Nanovskaya et al. studied the effect of the efflux transporter P-glycoprotein in the transplacental distribution of methadone and demonstrated the ability of this transporter to excrete methadone from the placental tissue back into the maternal circulation (19 ), further suggesting its involvement in protecting the fetus from xenobiotics.
Our comparison of methadone and EDDP concentrations in placenta and matched meconium samples revealed differences between these matrices in the distribution of these analytes. Methadone and EDDP concentrations were much lower in placenta than in meconium: median meconium/placenta methadone ratio, 4.8 (range, 0.2–13.6); median meconium/placenta EDDP ratio, 293.2 (range, 88.9 – 801.3). Furthermore, the 2 matrices differed with respect to the relative abundance of methadone and EDDP [mean (SD) EDDP/methadone concentration ratio, 0.11 (0.06) in placenta and 11.8 (17.2) in meconium]. These differences may be explained by fetal methadone metabolism and subsequent EDDP accumulation in meconium. Several cytochrome P450 (CYP) isoenzymes were identified in fetal liver, with CYP3A7 accounting for up to 50% of the CYP content in the fetal liver (20 ). CYP3A7 production decreases during gestation, with metabolism shifting within days of birth to CYP3A4 (20, 21 ), which is, in conjunction with CYP2B6, the principal enzyme for methadone biotransformation (22 ).
In this study, we also evaluated the possible correlations between methadone and EDDP concentrations in placenta, maternal methadone doses, and neonatal outcomes. Placental methadone and EDDP concentrations were significantly correlated, with methadone concentrations far exceeding EDDP concentrations, as is found in maternal plasma (plasma EDDP/methadone concentration ratios, 0.06 – 0.18) (12, 15 ). Placental EDDP appears to derive primarily from EDDP in the maternal plasma, because perfused placental tissue converts <1% of methadone to EDDP (16 ). To our knowledge, we present the first data for placental methadone and EDDP concentrations and their correlation with maternal methadone dose. Collection of plasma samples in the last weeks of pregnancy, if medically acceptable, might have been useful to evaluate the correlations between maternal methadone dose and plasma methadone and EDDP concentrations, and between plasma and placental concentrations. Plasma was collected only when medically necessary, however. Thus, an assessment of this correlation was not possible for our cohort. Nevertheless, in support of our findings of significant correlations between placental methadone concentrations and maternal methadone dose, other authors have also observed a strong correlation between the methadone concentration in umbilical cord blood and the maternal methadone dose (r = 0.768; P < 0.0001) (23 ). The relationships between methadone dose and concentration reported for maternal plasma have been inconsistent, however (13, 23–25 ).
Placenta EDDP concentration was negatively correlated with newborn head circumference (r = −0.579; P = 0.009). The mechanism responsible for this correlation is not clear, because exposure to the active methadone component is thought to be the agent responsible for this outcome. A possible explanation is that higher EDDP concentrations simply reflect higher methadone concentrations, followed by metabolism to the inactive metabolite. Furthermore, the placenta metabolizes methadone poorly, as in vitro studies have suggested (16 ); thus, the majority of EDDP in placenta appears to derive from maternal methadone metabolism. The clinical significance of this finding is unknown and requires further investigation. We also noted a positive correlation between the placental EDDP/methadone concentration ratio and the peak NAS score. Our results suggest that the placenta concentration might predict NAS intensity and therefore a need for neonatal treatment. The sample size in our study was small, however, and the correlation values (r = 0.513) indicate that the clinical relevance of this association is uncertain and requires further research. In support of our findings, previous studies have shown NAS of higher intensity to be associated with lower neonatal plasma methadone concentrations (23 ), a greater rate of decline in neonatal methadone concentration (13 ), or both (26 ). Mack et al. (25 ), however, found no correlation between the methadone concentration in the plasma of newborns and the intensity of withdrawal symptoms.
Meconium is considered the matrix of choice to detect in utero drug exposure, and assays of meconium samples primarily detect drug consumption in the third trimester (27 ). Placenta may be an effective alternative, however, if meconium is unavailable because of fetal distress and in utero discharge (28 ). The main advantage of placenta is its easy and noninvasive collection at birth. Only a few reports on the concentrations of illicit drugs in the placenta, however, are available to date (29 –33 ), and the window of drug detection has not been defined.
We also analyzed placenta samples for cocaine and opiates. The availability of cocaine and opiate results for matched meconium and urine samples collected throughout pregnancy allowed us to compare the usefulness of these matrices for detecting drug exposure in utero. The results for urine samples confirmed maternal opiate and cocaine consumption in the second and third trimesters in 17 and 9 participants, respectively. A lower urinary BE cutoff concentration of 150 μg/L, proposed for the 2008 revision of the Mandatory Guidelines for Federal Workplace Drug Testing Programs (34 ) may have increased cocaine detection. Among our participants, only 1 placenta sample contained opiates and cocaine; unfortunately, the unavailability of complete urine data for this participant precluded an evaluation of the placenta’s window of drug detection. Our results show that the placental window for detecting cocaine and opiates is quite short, given that several of the women used these drugs within a few weeks of delivery and that this drug use was not reflected in the placental samples. Moreover, the placental window appears to be much shorter than for meconium, for which positive results were found, even when the last day of cocaine and/or opiate intake was during the second trimester. Although the inclusion of other metabolites (e.g., mOHBE) in the analytical panel for placenta may increase the detection of drug exposure, our data show that meconium and regular urine collection provide more information than placental analysis. Meconium clearly is the preferred matrix for detecting in utero drug exposure. Placenta collection for drug testing would be justifiable only if meconium were unavailable. These preliminary data do suggest, however, that placenta methadone and EDDP concentrations could be valuable if meconium is unavailable and if recent drug exposure is forensically important.
Footnotes
Nonstandard abbreviations: EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; NAS, neonatal abstinence syndrome; JHBMC, Johns Hopkins Bayview Medical Center; BE, benzoylecgonine; mOHBE, m-hydroxybenzoylecgonine; CYP, cytochrome P450.
An abstract on this topic was presented at the International Association of Forensic Toxicology annual meeting in Bonn, Germany (August 29 to September 2, 2010).
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: R.E. Johnson, Reckitt Benckiser Pharmaceuticals.Consultant or Advisory Role: None declared.
Stock Ownership: R.E. Johnson, Reckitt Benckiser Pharmaceuticals (immediate family member).
Honoraria: None declared.
Research Funding: Intramural Research Program, and Extramural Grants DA12220 and DA12403 from the National Institute on Drug Abuse, NIH.
Expert Testimony: None declared.
Other Remuneration: A. de Castro, Dirección Xeral de Investigacion, Desenvolvemento e Innovación, Consellería de Innovación e Industria (AA-053), Galicia, Spain.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
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