The Relationship between methylation and genes associated with opioid response in humans.

Opioids are used to alleviate pain however ~10–30% of the Caucasian population obtain ineffective pain relief and / or intolerable side effects. Genetic polymorphisms in opioid pharmacodynamic and pharmacokinetic important genes have been investigated however there has been a lack of conclusive results. Epigenetic gene regulation is another study field of interest. DNA methylation is a gene regulatory mechanism that has been associated with gene repression or expression. Thus it was hypothesised that variable opioid response was influenced by methylation alterations. The promoter region methylation of ABCB1/MDR1, CYP2D6 and OPRM1 genes were analysed by pyrosequencing in smoking, opioid exposed and control pilot populations. Genetic variations within ABCB1/MDR1, COMT, CYP2B6, CYP2D6 and OPRM1 genes were also investigated. Tissue opioid concentrations were determined by HPLCMS/ MS and GC-MS/MS. DNA methylation was influenced by chronic opioid exposure and / or the lifestyle associated with opioid dependency, but not by cigarette smoke exposure. An increase in DNA methylation was observed in the opioid exposed baby population but this was not indicative of development of neonatal abstinence syndrome (NAS). Associations between the CYP2B6*6 genotype and NAS development were found. No relationship was observed between gene methylation and opioid response but variants in OPRM1 and ABCB1/MDR1 exhibited a relationship with opioid response in cancer pain. This investigative pilot research revealed that some genetic variants can be used as diagnostic markers to predict susceptibility of methadone exposed babies to NAS development, and others are associated with variable opioid response in a cancer patient population. Although DNA methylation was not observed to be a contributory factor to opioid response, this research has ascertained the influence of chronic opioid exposure on DNA methylation of various biological samples. This finding is significant for future studies

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

BackgroundThis 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.MethodsLiver, 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.ResultsLiver 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.ConclusionsThere 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

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

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

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 degrees C and -18 degrees 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 degrees C and -18 degrees C. Cocaine stability in human muscle obtained from cocaine positive forensic cases was assessed following storage at -18 degrees 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 degrees 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 degrees C and had declined by 81% following storage at -18 degrees C. At 4 degrees 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 degrees C was essentially stable for the study period whereas at 4 degrees C concentrations decreased by &gt;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 degrees 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 degrees 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. (C) 2011 Elsevier Ireland Ltd. All rights reserved.Bournemouth UniversityBournemouth UniversityFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), BrazilFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Brazil [2010/11517-6

Real-time near-body drug screening during autopsy I:use of the Randox biochip drugs of abuse DOA I and DOA II immunoassays

Screening for drugs of abuse is widely employed as part of forensic toxicology investigations. The nature of specimens collected at autopsy varies depending on local customs, the case circumstances, and the condition of the cadaver. It is generally accepted that wherever possible specimens of peripheral blood, liver, stomach contents, vitreous humor, and muscle can be useful for toxicological analysis. In some countries, legislation or religious custom may mitigate against the removal of postmortem tissue unless shown to be necessary. The availability of a sensitive and broad ranging near-body screening test may provide a useful tool to assist pathologists in making decisions regarding the retention of tissues for toxicology analysis. We describe the performance of the Randox drugs-of-abuse (DOA) arrays, DOA I and DOA II, for near-body screening using whole blood, urine, vitreous humor, liver, and psoas major muscle. Samples were obtained from 106 autopsies and screened for the presence of amphetamine, barbiturates, benzodiazepines, benzoylecgonine, buprenorphine, cannabinoids, fentanyl, ketamine, lysergic acid diethylamide, methadone, methamphetamine, methaqualone, methylenedioxymethylamphetamine (Ecstasy), opiates, oxycodone, phencyclidine, and propoxyphene in the mortuary whilst the postmortem was being performed. Blood from each case underwent confirmatory analysis using either gas chromatography-mass spectrometry, liquid chromatography with tandem mass spectrometry or diode array detection. Excellent agreement between the near-body screening tests on a variety of tissues and confirmatory analyses in blood was obtaine