Increased DNA Methylation of ABCB1, CYP2D6, and OPRM1 Genes in Newborn Infants of Methadone-Maintained Opioid-Dependent Mothers.

OBJECTIVE: To investigate whether in utero opioid exposure, which has been linked to adverse neurodevelopmental and social outcomes, is associated with altered DNA methylation of opioid-related genes at birth. STUDY DESIGN: Observational cohort study of 21 healthy methadone-maintained opioid-dependent mother-infant dyads consecutively delivered at >36 weeks of gestation, and 2 comparator groups: smoking, "deprived" opioid-naïve mother-infant dyads (n = 17) and nonsmoking, "affluent" opioid-naïve mother-infant dyads (n = 15). DNA methylation of ABCB1, CYP2D6, and OPRM1 genes for mothers and babies was determined from buccal swabs. Plasma methadone concentrations were additionally measured for methadone-maintained opioid-dependent mothers. RESULTS: DNA methylation for ABCB1 and CYP2D6 was similar in opioid-naïve infants compared with their mothers, but was less for OPRM1 (3 ± 1.6% vs 8 ± 1%, P < .0005). Opioid-exposed newborns had similar DNA methylation to their mothers for all genes studied and greater methylation of ABCB1 (18 ± 4.8% vs 3 ± 0.5%), CYP2D6 (92 ± 1.2% vs 89 ± 2.4%), and OPRM1 (8 ± 0.3% vs 3 ± 1.6%) compared with opioid-naïve newborns (P < .0005 for all 3 genes). Infant DNA methylation was not related to birth weight, length of hospital stay, maternal smoking, dose or plasma concentration of methadone at delivery, or postcode of residence. CONCLUSIONS: In utero exposure to opioids is associated with increased methylation of opioid-related genes in the newborn infant. It is not clear whether these findings are due to opioid exposure per se or other associated lifestyle factors.

Variations in Infant CYP2B6 Genotype Associated with the Need for Pharmacological Treatment for Neonatal Abstinence Syndrome in Infants of Methadone-Maintained Opioid-Dependent Mothers.

Background Neonatal abstinence syndrome (NAS) in infants of methadone-maintained opioid-dependent (MMOD) mothers cannot be predicted in individual cases. We investigated whether variation in infant genotype is associated with severity of NAS. Methods This is a pilot observational cohort study of 21 MMOD mothers and their newborns. Infant buccal swabs were obtained soon after delivery, together with a maternal blood sample for the determination of maternal plasma methadone concentration. Genomic variation in five opioid-related genes (ABCB1, COMT, CYP2B6, CYP2D6, and OPRM1) was ascertained from infant buccal swabs and related to need for pharmacological treatment of NAS. Results Out of 21 infants, 11 (52%) required treatment for NAS. Mothers of treated infants tended to have been prescribed higher doses of methadone, but plasma methadone concentrations did not differ between mothers of treated or untreated babies. Treated and untreated babies did not differ in terms of method of feeding. Treated infants were more likely to carry the normal (homozygous) allele at 516 and 785 regions of CYP2B6 gene (p = 0.015 and 0.023, respectively). There were no differences in any other genes between infants who did or did not require treatment for NAS. Conclusion Genomic variation in CYP2B6 may explain, at least in part, severity of NAS.

Use of the Randox Evidence Investigator immunoassay system for near-body drug screening during post-mortem examination in 261 forensic cases.

BACKGROUND: This paper describes the performance of four Randox drug arrays, designed for whole blood, for the near-body analysis of drugs in a range of post-mortem body specimens. METHODS: Liver, psoas muscle, femoral blood, vitreous humor and urine from 261 post-mortem cases were screened in the mortuary and results were obtained within the time taken to complete a post-mortem. Specimens were screened for the presence of amfetamine, barbiturates, benzodiazepines, benzoylecgonine, buprenorphine, cannabinoids, dextropropoxyphene, fentanyl, ketamine, lysergide, methadone, metamfetamine, methaqualone, 3,4-methylenedioxymetamfetamine, opioids, paracetamol, phencyclidine, salicylate, salicylic acid, zaleplon, zopiclone and zolpidem using the DOA I, DOA I+, DOA II and Custom arrays. RESULTS: Liver and muscle specimens were obtained from each of the 261 post-mortem cases; femoral blood, vitreous humor and urine were available in 98%, 92% and 72% of the cases, respectively. As such, the equivalent of 12,978 individual drug-specific, or drug-group, immunoassay tests were undertaken. Overall >98% of the 12,978 screening tests undertaken agreed with laboratory confirmatory tests performed on femoral blood. CONCLUSIONS: There is growing interest in the development of non-invasive procedures for determining the cause of death using MRI and CT scanning however these procedures are, in most cases, unable to determine whether death may have been associated with drug use. The Randox arrays can provide qualitative and semi-quantitative results in a mortuary environment enabling pathologists to decide whether to remove specimens from the body and submit them for laboratory analysis. Analysis can be undertaken on a range of autopsy specimens which is particularly useful when conventional specimens such as blood are unavailable.

Validation of a gas chromatography-ion trap-tandem mass spectrometry assay for the simultaneous quantification of cocaine, benzoylecgonine, cocaethylene, morphine, codeine, and 6-acetylmorphine in aqueous solution, blood, and skeletal muscle tissue.

A gas chromatography-ion trap-tandem mass spectrometry method was developed and validated for the simultaneous solid-phase extraction and quantification of cocaine, benzoylecgonine, cocaethylene, morphine, codeine, and 6-monoacteylmorphine in blood, muscle tissue, and water. An assay for total morphine in blood and muscle was also validated. The limit of quantification was ≤ 0.01 mg/kg in muscle and ≤ 0.005 mg/L in blood and water. Good linearity was observed over the concentration ranges studied (r ≥ 0.99). The repeatability (% RSD) at the three concentration levels was typically ≤ 15% and never exceeded 17%. Intermediate precision of ≤ 16% was obtained for all matrices. Deviation < 20% of the nominal concentration was obtained in all repeatability and intermediate precision experiments. Extraction recoveries for cocaine and metabolites ranged from 91% to 110% in water, 81% to 110% in blood, and 61% to 75% in muscle. Recoveries for opiates ranged from 59% to 104% in water, 50% to 95% in blood, and 41% to 79% in muscle. The hydrolysis efficiency for the total morphine assay in blood and muscle ranged from 91% to 99% with within-day and intermediate precisions of ≤ 14% and ≤ 12%, respectively.

The effect of sodium fluoride preservative and storage temperature on the stability of 6-acetylmorphine in horse blood, sheep vitreous and deer muscle.

This study examined the in vitro stability of 6-acetylmorphine (6AM) in horse blood, sheep vitreous humour (VH) and homogenised deer muscle stored under different storage conditions. The stability of 6AM in horse blood is of interest because many toxicological laboratories utilise this matrix for the preparation of blood calibration and check standards and the latter are typically stored during routine use. Data on the storage stability of 6AM in human VH is extremely limited and no data has been reported in muscle. In the absence of human samples, 6AM stability was demonstrated in sheep vitreous and deer muscle. Blood and VH were stored with and without NaF at room temperature (RT), 4 and -18°C for 84 days. Muscle tissue homogenates were prepared in water with and without NaF and also in phosphate buffer (pH 6.0) containing NaF. Homogenates were stored for 31 days at RT, 4 and -18°C. Morphine and 6AM were extracted using SPE and quantified by GC-ion trap-MS/MS. In the absence of NaF, 6AM could not be detected after 7 and 14 days in blood stored at RT and 4°C, respectively. Although at -18°C 6AM was stable for 7 days (12% loss), only 54% was detected by day 84. The addition of NaF to horse blood increased 6AM stability substantially at every temperature. Further, the rate of degradation was found to be significantly slower in blood preserved with 2% NaF compared with 1% NaF (p=.05). 6AM was stable for the study period in preserved blood (1 and 2% NaF) stored at -18°C. For laboratories utilising horse blood in the preparation of standards, preservation with 1% NaF (minimum) and storage at -18°C is recommended. The addition of NaF to VH was essential for 6AM stability. Irrespective of temperature substantial losses (≥ 42%) were observed in unpreserved sheep VH by day 7. In preserved VH the concentration declined by only 22% on day 7 following storage at RT and no loss observed in VH stored at 4 and -18°C at the same time. In muscle, 6AM was stable for 7 days in preserved samples stored at RT and in all samples stored at 4°C and below. The addition of NaF increased the stability of 6AM substantially in muscle. The increased stability of 6AM in VH and muscle preserved with fluoride was attributed to inhibition of bacterial action and the subsequent reduction in the rate of putrefaction of these tissues.

The effect of sodium fluoride preservative and storage temperature on the stability of cocaine in horse blood, sheep vitreous and deer muscle.

The in vitro stability of cocaine in horse blood, sheep vitreous humour (VH) and homogenised deer muscle is described. The stability of cocaine in horse blood was of interest because many toxicology laboratories utilise horse blood for the preparation of calibration and check standards and the latter are typically stored during routine use. The storage stability of cocaine in human VH and muscle has not been previously reported. In the absence of blank human VH and muscle, cocaine stability under varying conditions was demonstrated in animal tissues. Blood and VH were stored with and without addition of NaF at room temperature (RT), 4°C and -18°C for 84 days. Muscle homogenates were prepared in water, water/2% NaF, and phosphate buffer (pH 6.0)/2% NaF, and stored for 31 days at RT, 4°C and -18°C. Cocaine stability in human muscle obtained from cocaine positive forensic cases was assessed following storage at -18°C for 13 months. Cocaine and benzoylecgonine (BZE) were extracted using SPE and quantified by GC-MS/MS. Cocaine was stable for 7 days in refrigerated (4°C) horse blood fortified with 1 and 2% NaF. In the absence of NaF, cocaine was not detectable by day 7 in blood stored at RT and 4°C and had declined by 81% following storage at -18°C. At 4°C the rate of cocaine degradation in blood preserved with 2% NaF was significantly slower than with 1% NaF. The stability of cocaine in horse blood appeared to be less than that reported for human blood, probably attributable to the presence of carboxylesterase in horse plasma. Cocaine stored in VH at -18°C was essentially stable for the study period whereas at 4°C concentrations decreased by >50% in preserved and unpreserved VH stored for longer than 14 days. Fluoride did not significantly affect cocaine stability in VH. The stability of cocaine in muscle tissue homogenates significantly exceeded that in blood and VH at every temperature. In preserved and unpreserved samples stored at 4°C and below, cocaine loss did not exceed 2%. The increased stability of cocaine in muscle was attributed to the low initial pH of post-mortem muscle. In tissue from one human case stored for 13 months at -18°C the muscle cocaine concentration declined by only 15% (range: 5-22%). These findings promote the use of human muscle as a toxicological specimen in which cocaine may be detected for longer compared with blood or VH.