Mimi Stokke Opdal

Genetic Variation of VKORC1 and CYP4F2 Genes Related to Warfarin Maintenance Dose in Patients with Myocardial Infarction

The aim of this study was to investigate whether the VKORC1*3 (rs7294/9041 G > A), VKORC1*4 (rs17708472/6009 C > T), and CYP4F2 (rs2108622/1347 C > T) polymorphisms were associated with elevated warfarin maintenance dose requirements in patients with myocardial infarction (n = 105) from the Warfarin Aspirin Reinfarction Study (WARIS-II). We found significant associations between elevated warfarin dose requirements and VKORC1*3 and VKORC1*4 polymorphisms (P = .001 and P = .004, resp.), whereas CYP4F2 (1347 C > T) showed a weak association on higher warfarin dose requirements (P = .09). However, analysing these variant alleles in a regression analysis together with our previously reported data on VKORC1*2, CYP2C9*2 and CYP2C9*3 polymorphisms, gave no significant associations for neither VKORC1*3, VKORC1*4 nor CYP4F2 (1347 C > T). In conclusion, in patients with myocardial infarction, the individual contribution to warfarin dose requirements from VKORC1*3, VKORC1*4, and CYP4F2 (1347 C > T) polymorphisms was negligible. Our results indicate that pharmacogenetic testing for VKORC1*2, CYP2C9*2 and CYP2C9*3 is more informative regarding warfarin dose requirements than testing for VKORC1*3, VKORC1*4, and CYP4F2 (1347 C > T) polymorphisms.

Blood GHB concentrations and results of medical examinations in 25 car drivers in Norway

PurposeInformation on the clinical effects associated with whole blood gamma-hydroxybutyrate (GHB) concentrations is sparse. We have investigated possible relationships between GHB blood concentrations and clinical effects in car drivers.MethodsIn Norway, the police stop car drivers suspected of drug-driving. Medical doctors perform a clinical test of impairment (CTI) and blood samples are screened for drugs/medicines by immunological, enzymatic and chromatographic methods at the Division of Forensic Toxicology and Drug Abuse. GHB is a part of our extended drug-testing programme. GHB is standardly measured as GBL by gas chromatographic method. All the results were stored in a database. This database was searched between 2000 and 2007 for car drivers positive only for GHB, called GHB-drivers. A control group with a completely negative blood analysis, including GHB, called control-drivers, was included in the study.ResultsTwenty-five car drivers had only GHB in their blood. The police reported that 78% showed unsafe driving behaviour and seven were involved in car accidents, without serious injury. A total of 61% of the drivers were found to be sleepy or in an even more reduced state of consciousness. The median GHB blood concentration was 1,262 (range 592–2,191) μmol/L, measured a median of 69 min after the police had stopped the driver from driving. The GHB blood concentration tended to increase with increasing impairment and reduced consciousness. Clinical findings were normal- to large-sized pupils (86%), impairment as the final conclusion (84%), impaired balance/nystagmus (62 and 54%, respectively), congested/shiny conjunctiva (67%), apathetic, aggressive or abnormal behaviour (65%), reduced short-term memory (67%), reduced/absent pupillar reaction to light (65%), heart rate ≤ 70 beats/min (56%), and some level of reduced consciousness (56%). In the control-drivers, 15.6% were found by the medical doctors to have reduced consciousness or impaired.ConclusionsThe median GHB blood concentration of the 25 car drivers was high. Most drivers had clinical impairment that was not explainable by injuries, with depressive effects on the central nervous system and sympathomimetic effects on eyes. Effects on impairment and consciousness tended to be concentration-dependent. The number of drivers who were impaired or had reduced consciousness was highly increased in GHB-drivers compared to controls. Based on these results, we conclude that the GHB-drivers most probably drove in an unsafe manner due to impairment by GHB.

Relationship between methadone and EDDP (2 -ethylidene -1,5 -dimethyl -3,3 -diphenylpyrrolidine) in urine samples from Norwegian prisons

BackgroundMethadone maintenance treatment is a widely used therapy in the rehabilitation of opioid addiction the world over. Methadone is metabolised in the body to a number of inactive metabolites, but primarily to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). The Division of Forensic Toxicology and Drug Abuse (DFTDA) of the Norwegian Institute of Public Health carries out drug analysis of urine samples from inmates of prisons throughout Norway. Methadone and EDDP in the urine are also tested for upon request. The results are stored in a secure database at the DFTDA.ObjectivesThe aims of the present study were (1) to observe variations in methadone and EDDP concentrations in urine in relation to urine pH in a large set of urine samples obtained from prison inmates and (2) to analyse samples testing methadone-positive/EDDP-negative and investigate whether such results could occur naturally, without sample tampering.MethodsAll urine samples that tested positive for methadone over the period 2004–2005 were collected from the DFTDA database, and the relation between methadone and EDDP excretion in urine, and urinary pH was determined. Samples that tested positive for methadone but negative for EDDP were picked out and studied individually.ResultsA total of 1539 urine samples (cases) had tested positive for methadone in our database for the period 2004–2005. There was a strong correlation between the concentration of methadone in urine and urine pH in these samples, with higher concentrations of methadone present at lower pH levels. Cases that tested positive for methadone but negative for EDDP were rare – a total of five (0.3% of all cases tested). These cases were studied in more detail.Conclusion Methadone excretion in urine is dependent on urinary pH. Methadone-positive/EDDP-negative results may suggest sample tampering in some, but not all, cases.

Blood Alcohol Concentrations in Apprehended Drivers of Cars and Boats Suspected to Be Impaired by the Police

Objective. According to the Norwegian Road Traffic Act, car drivers are not allowed to operate a vehicle with a blood alcohol concentration (BAC) above 0.2 g/kg. Depending on the size of the boat or ship, boat drivers/captains/first mates are not allowed to conduct the boat with a BAC above 0.8 g/kg when driving small boats (length less than 15 m) and above 1.5 g/kg when running larger vessels/ships. The new Sea Act of June 2005 states that captains/first mates cannot conduct a ship if he/she has a BAC above 0.2 g/kg. Our aim was to determine the current median BAC in a large population of car and boat drivers in Norway. Our other aim was to study if median BAC was higher in boat drivers than in car drivers who were suspected by the police to be impaired. Furthermore, we wanted to investigate if the BAC levels were differently distributed by gender or age within and between these two groups. Methods. The Norwegian Institute of Public Health analyzes blood samples from all car/boat drivers suspected of driving under the influence of alcohol and non-alcoholic drugs. In the present study, samples submitted between 01.05 and 01.09 in 2002–2004 were included. Drivers, who in addition tested positive for drugs or abuse substances other than ethanol were excluded. Results. There were 321 boat drivers and 3,061 car drivers who were suspected to be under the influence of ethanol only. The median BAC in boat drivers (1.76 g/kg [range 0.02–3.54]) was significantly higher compared to that in car drivers (1.54 g/kg [range 0.00–4.27]). In the car driver group, the mean BAC did not differ significantly between men and women. The median level of BAC was significantly higher in men than in women in the boat driver group (1.77 g/kg with CI 1.69–1.85 vs. 1.27 g/kg with CI 0.78–1.76). Conclusions. Alcohol impairment of car drivers is known to be considered the most important contributing cause of car crash injuries. Driving a boat may demand the same degree of performance skills as driving a car. The median BAC in apprehended boat drivers was considerably high in the present study. The median BAC was also high in car drivers despite strict legislation. The population of drivers of cars in our study, however, is from previous studies known to contain a large proportion of heavy drinkers. Less is known about the drinking habits in boat drivers, and caution is needed in generalizing from our results. However, our results indicate the possible need for stricter legislation and more frequent police control that will hopefully prevent serious accidents caused by ethanol drinking at sea.

Therapeutic Drug Monitoring of Clobazam and Its Metabolite-Impact of Age and Comedication on Pharmacokinetic Variability.

Background: Clobazam (CLB) has been used as an antiepileptic drug for several decades. There is still insufficient data regarding its pharmacokinetic variability in clinical practice. The purpose of this study was to investigate pharmacokinetic variability of CLB with emphasis on the impact of age and comedication in patients with epilepsy. Methods: Serum concentration measurements of CLB and its metabolite N-desmethylclobazam (NCLB), as well as demographic and clinical data were retrieved from the routine therapeutic drug monitoring service at the National Center for Epilepsy, Norway, 2009–2013. NCLB/CLB and total (CLB + NCLB), CLB and NCLB concentration/dose (C/D) ratios were calculated. Results: 550 patients (296 women/254 men), average age 27 years (range 1–86), were included. The interindividual pharmacokinetic variability was extensive, as illustrated by a 100-fold variability in serum concentration compared with dose (total C/D ratio 0.03–3.29 µmol·L−1·mg−1). The CLB C/D ratio was 36% lower in young children (2–9 years) than in adults (18–64 years), reflecting a higher clearance. In patients receiving phenytoin, felbamate, stiripentol, oxcarbazepine or eslicarbazepine acetate, valproate, phenobarbital, zonisamide or carbamazepine one or more of the calculated ratios were significantly different from that in patients receiving no or neutral comedications. The mean values for the different groups were in the order of 20%–230% of C/D ratios in the neutral group and 200%–950% of the NCLB/CLB ratio. Conclusions: The pharmacokinetic variability of CLB and its metabolite NCLB in clinical practice is extensive, and is influenced by drug–drug interactions, age, and pharmacogenetics. Therapeutic drug monitoring of CLB and NCLB is therefore valuable in patient management.

Serum bilirubin concentration in healthy adult North-Europeans is strictly controlled by the UGT1A1 TA-repeat variants.

The major enzyme responsible for the glucuronidation of bilirubin is the uridine 5′-diphosphoglucose glucuronosyltransferase A1 (UGT1A1) enzyme, and genetic variation in the UGT1A1 gene is reported to influence the bilirubin concentration in the blood. In this study, we have investigated which gene-/haplotype variants may be useful for genetic testing of Gilberts syndrome. Two groups of samples based on serum bilirubin concentrations were obtained from the Nordic Reference Interval Project Bio-bank and Database (NOBIDA): the 150 individuals with the highest bilirubin (>17.5 µmol/L) and the 150 individuals with normal bilirubin concentrations (<17.5 µmol/L). The individuals were examined for the TA6>TA7 variant in the UGT1A1 promoter and 7 tag-SNPs in an extended promoter region of UGT1A1 (haplotype analysis) and in selected SNPs in candidate genes (SLCO1B3, ABCC2 and NUP153). We found significant odds ratios for high bilirubin level for all the selected UGT1A1 variants. However, in stepwise multivariate logistic regression analysis of all genetic variants together with age, sex, country of origin and fasting time, the repeat variants of UGT1A1 TA6>TA7 and SLCO1B3 rs2117032 T>C were the only variants significantly associated with higher bilirubin concentrations. Most individuals with high bilirubin levels were homozygous for the TA7-repeat (74%) while only 3% were homozygous for the TA7-repeat in individuals with normal bilirubin levels. Among individuals heterozygous for the TA7-repeat, a low frequent UGT1A1-diplotype harboring the rs7564935 G-variant was associated with higher bilirubin levels. In conclusion, our results demonstrate that in testing for Gilberts syndrome, analyzing for the homozygous TA7/TA7-genotype would be appropriate.

Effects of Hemodialysis on Methadone Pharmacokinetics and QTc

PURPOSE Effects of hemodialysis on pharmacokinetic properties and QTc were studied in 4 patients taking daily methadone dose of 100 mg (range, 60-120 mg). METHODS Methadone in serum, dialysate, and urine were measured by LC-MS/MS. QTc was calculated with Bazetts formula. FINDINGS The serum Cmin methadone level was 1124 nmol/L (range, 547-1581 nmol/L). Methadone dialysate clearance was 17.1 mL/min (range, 13.7-20.6 mL/min). Total loss in dialysate was 2.30% (range, 1,25-3,70%) of daily methadone intake. QTc increased from 391 msec (range, 369-406 msec) to 445 msec (range, 407-479 msec), independently of serum methadone level, which may be explained by normalization of serum electrolytes. IMPLICATIONS Methadone dose adjustment is not needed because of hemodialysis.

Developing a rapid semi-automated sample preparation with alkaline hydrolysis in a 96-well plate for quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine samples by UHPLC-MS/MS

HighlightsSemi‐automated sample preparation of 11‐nor‐&Dgr;9‐tetrahydrocannabinol‐9‐carboxylic acid (THCA) in 96 well plate format with UHPLC‐MS/MS.Hydrolysis of THCA‐glucuronide in a 96‐well plate.A wide linear calibration curve from 5 to 4000 ng/ml.Precautions to minimize the surface adsorption of THCA.Comparing the results of manual sample preparation with semi‐automatic sample preparation in different type of 96‐well plates. ABSTRACT A simple, sensitive, rapid and robust manual (in glass tubes) and semi‐automatic method (in a 96‐well plate format) for quantification of 11‐nor‐&Dgr;9‐tetrahydrocannabinol‐9‐carboxylic acid (THCA) in urine using a UHPLC‐MS/MS have been developed. The methods involved hydrolysis with potassium hydroxide in a 96‐well plate, followed by neutralization and dilution of the sample in one step with a stable solution of formic acid‐methanol‐acetonitrile‐water (2.2%/35/35/30) before centrifugation. The total chromatographic run time was 4.9 min, with retention times of 1.6 min for THCA. The linear calibration range (5–4000 ng/ml) prepared in urine matrix was wide to avoid reanalyzing. The methods take especially precautions to totally eliminate the carry over after run of the highest standard and to eliminate adsorption of THCA on lab‐ware during sample preparation. High content of organic solvent in the neutralization and dilution solution and in the initial composition of the mobile phase gradient are therefore necessary. Standards and quality controls was pH adjusted to 8.4 to increase the solubility of the hydrophobic THCA in urine and prevent adsorption during storage. A preliminary study revealed that adsorption of THCA during semi‐ automatic sample preparation in a 96‐well plate (plastic) was less compared to manual sample preparation in glass tube. The intermediate precision and accuracy by the manual and semi‐automated method were less than 10% and ranged from 88% to 114% respectively. Results from external controls from a proficiency testing programs were within 20% accuracy. 63 urine samples positive by immunoassay screening of cannabinoids were confirmed by UHPLC‐MS/MS using routine manual preparation in glass tubes and semi‐automatic sample preparation using 96‐well plastic and glass coated plate. Percentage difference of the measurements were calculated and plotted according to Bland & Altman and the mean percentage difference was not significant.