A DNA methylation biomarker of alcohol consumption.

The lack of reliable measures of alcohol intake is a major obstacle to the diagnosis and treatment of alcohol-related diseases. Epigenetic modifications such as DNA methylation may provide novel biomarkers of alcohol use. To examine this possibility, we performed an epigenome-wide association study of methylation of cytosine-phosphate-guanine dinucleotide (CpG) sites in relation to alcohol intake in 13 population-based cohorts (ntotal=13 317; 54% women; mean age across cohorts 42-76 years) using whole blood (9643 European and 2423 African ancestries) or monocyte-derived DNA (588 European, 263 African and 400 Hispanic ancestry) samples. We performed meta-analysis and variable selection in whole-blood samples of people of European ancestry (n=6926) and identified 144 CpGs that provided substantial discrimination (area under the curve=0.90-0.99) for current heavy alcohol intake (⩾42 g per day in men and ⩾28 g per day in women) in four replication cohorts. The ancestry-stratified meta-analysis in whole blood identified 328 (9643 European ancestry samples) and 165 (2423 African ancestry samples) alcohol-related CpGs at Bonferroni-adjusted P<1 × 10-7. Analysis of the monocyte-derived DNA (n=1251) identified 62 alcohol-related CpGs at P<1 × 10-7. In whole-blood samples of people of European ancestry, we detected differential methylation in two neurotransmitter receptor genes, the γ-Aminobutyric acid-A receptor delta and γ-aminobutyric acid B receptor subunit 1; their differential methylation was associated with expression levels of a number of genes involved in immune function. In conclusion, we have identified a robust alcohol-related DNA methylation signature and shown the potential utility of DNA methylation as a clinically useful diagnostic test to detect current heavy alcohol consumption.

An epigenome-wide association study meta-analysis of educational attainment.

The epigenome is associated with biological factors, such as disease status, and environmental factors, such as smoking, alcohol consumption and body mass index. Although there is a widespread perception that environmental influences on the epigenome are pervasive and profound, there has been little evidence to date in humans with respect to environmental factors that are biologically distal. Here we provide evidence on the associations between epigenetic modifications-in our case, CpG methylation-and educational attainment (EA), a biologically distal environmental factor that is arguably among the most important life-shaping experiences for individuals. Specifically, we report the results of an epigenome-wide association study meta-analysis of EA based on data from 27 cohort studies with a total of 10 767 individuals. We find nine CpG probes significantly associated with EA. However, robustness analyses show that all nine probes have previously been found to be associated with smoking. Only two associations remain when we perform a sensitivity analysis in the subset of never-smokers, and these two probes are known to be strongly associated with maternal smoking during pregnancy, and thus their association with EA could be due to correlation between EA and maternal smoking. Moreover, the effect sizes of the associations with EA are far smaller than the known associations with the biologically proximal environmental factors alcohol consumption, body mass index, smoking and maternal smoking during pregnancy. Follow-up analyses that combine the effects of many probes also point to small methylation associations with EA that are highly correlated with the combined effects of smoking. If our findings regarding EA can be generalized to other biologically distal environmental factors, then they cast doubt on the hypothesis that such factors have large effects on the epigenome.

Dissecting the relationship between high-sensitivity serum C-reactive protein and increased fracture risk: the Rotterdam Study.

UNLABELLED: Serum high-sensitivity C-reactive protein (CRP) is an inflammatory biomarker. We investigated the relationship between CRP and bone health in the Rotterdam Study. Serum high-sensitivity CRP was associated with fracture risk and lower femoral neck bending strength. Mendelian randomization analyses did not yield evidence for this relationship being causal. INTRODUCTION: Inflammatory diseases are associated with bone pathology, reflected in a higher fracture risk. Serum high-sensitivity CRP is an inflammatory biomarker. We investigated the relationship between CRP and bone mineral density (BMD), hip bone geometry, and incident fractures in the Rotterdam Study, a prospective population-based cohort. METHODS: At baseline, serum high-sensitivity CRP was measured. A weighted genetic risk score was compiled for CRP based on published studies (29 polymorphisms; Illumina HumanHap550 Beadchip genotyping and HapMap imputation). Regression models were reported per standard deviation increase in CRP adjusted for sex, age, and BMI. Complete data was available for 6,386 participants, of whom 1,561 persons sustained a fracture (mean follow-up, 11.6 years). RESULTS: CRP was associated with a risk for any type of fracture [hazard ratio (HR) = 1.06; 95 % confidence interval (CI), 1.02-1.11], hip fractures (HR = 1.09; 1.02-1.17) and vertebral fractures [odds ratio (OR) = 1.34; 1.14-1.58]. An inverse relationship between CRP levels and section modulus (-0.011 cm(3); -0.020 to -0.003 cm(3)) was observed. The combined genetic risk score of CRP single nucleotide polymorphisms (SNPs) was associated with serum CRP levels (p = 9 × 10(-56)), but not with fracture risk (HR = 1.00; 0.99-1.00; p = 0.23). CONCLUSIONS: Serum high-sensitivity CRP is associated with fracture risk and lower bending strength. Mendelian randomization analyses did not yield evidence for this relationship being causal. Future studies might reveal what factors truly underlie the relationship between CRP and fracture risk.

Genetic variation may modify ovarian reserve in female childhood cancer survivors.

STUDY QUESTION: Are genetic polymorphisms, previously identified as being associated with age at menopause in the healthy population, associated with ovarian reserve and predicted age at menopause in adult long-term survivors of childhood cancer? SUMMARY ANSWER: The CT genotype of rs1172822 in the BRSK1 gene is associated with lower serum anti-Müllerian hormone (AMH) levels and a younger predicted age at menopause in adult survivors of childhood cancer. WHAT IS KNOWN ALREADY: Gonadotoxicity is a well-known late side effect of chemotherapy and radiotherapy in adult survivors of childhood cancer. In the healthy population, several genetic polymorphisms are associated with age at natural menopause. Currently, data on the impact of previously identified variants in gene loci associated with ovarian reserve in adult long-term survivors of childhood cancer are lacking. STUDY DESIGN, SIZE, DURATION: We performed a pilot study in a single-centre cohort of adult female Caucasian childhood cancer survivors (n = 176). PARTICIPANTS/MATERIALS, SETTING, METHODS: We determined serum AMH levels (a marker of ovarian reserve) in adult survivors of childhood cancer (n = 176) and studied single nucleotide polymorphisms (SNPs) previously reported to be associated with age at natural menopause: BRSK1 (rs1172822), ARHGEF7 (rs7333181), MCM8 (rs236114), PCSK1 (rs271924), IGF2R (rs9457827) and TNF (rs909253). Association analysis was performed using the additive genetic model. Linear regression was conducted to assess the effect of significant polymorphisms in two previously published menopause prediction models. MAIN RESULTS AND THE ROLE OF CHANCE: The CT genotype of rs1172822 in the BRSK1 (BR serine/threonine kinase 1) gene was negatively associated with serum AMH levels in our cohort (odds ratio: 3.15, 95% confidence interval: 1.35-7.32, P = 0.008) and significantly associated with the predicted age at menopause (P = 0.04). The other five SNPs were not associated with serum AMH levels. LIMITATIONS, REASONS FOR CAUTION: This is a pilot study showing preliminary data which must be confirmed. To confirm our findings and enlarge the project, a nationwide genome-wide association (GWA) project on the ovarian reserve in female survivors of childhood cancer should be performed, including a replication cohort. WIDER IMPLICATIONS OF THE FINDINGS: Our findings support the hypothesis that previously identified genetic polymorphisms associated with age at menopause in healthy women may have an effect on the onset of menopause in female survivors of childhood cancer. Our study highlights a new aspect of the influences on the ovarian reserve after childhood cancer, which should be investigated further in a nationwide GWA study. Eventually, this information can help us to improve counselling on fertility preservation prior to cancer treatment based on genetic factors in individual patients. STUDY FUNDING AND CONFLICT OF INTEREST: W.D. is supported by the Paediatric Oncology Centre Society for Research (KOCR), Rotterdam, The Netherlands. J.S.E.L. has received fees and grant support from the following companies (in alphabetic order): Ferring, Genovum, Merck-Serono, Organon, Schering Plough and Serono. All other authors have nothing to disclose.

The association between plasma homocysteine levels, methylation capacity and incident osteoporotic fractures.

BACKGROUND: An elevated level of plasma homocysteine (Hcy) is a known risk factor for osteoporotic fractures. In addition, Hcy is related to DNA-methylation metabolism. To determine whether the association between Hcy and fractures is explained by an altered methylation capacity, we investigated the associations between levels of s-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) and fracture risk. METHODS: We studied 503 females aged 55 years and over from the Rotterdam Study (RS) in whom plasma Hcy, SAM and SAH levels were measured. Bone mineral density (BMD) at the hip was assessed using DXA. Incident fractures were recorded over a mean period of 7.0 years. Cox proportional hazards analysis and linear regression were used to assess relationships between plasma metabolite levels, incident osteoporotic fractures and BMD. RESULTS: Over a total of 3502 person-years of follow-up, 103 subjects sustained at least one osteoporotic fracture. Whereas incidence of osteoporotic fractures was associated with quartiles of Hcy (p=0.047), it was not associated with quartiles of SAM, SAH or SAM/SAH-ratio (all p for trend>0.6). Stepwise linear regression showed that SAM/SAH-ratio, but not Hcy, was independently associated with hip BMD (ß=0.073, p=0.025). CONCLUSION: Since SAM, SAH and SAM/SAH-ratio were not associated with osteoporotic fractures, alterations in methylation capacity most likely do not appear to be an important factor in the association between Hcy and fractures.

Therapeutic drug monitoring of everolimus using the dried blood spot method in combination with liquid chromatography-mass spectrometry.

An assay of everolimus based on finger prick sampling and consecutive application as a blood spot on sampling paper has been developed. We explored several methods [K. Hoogtanders, J. van der Heijden, M. Christiaans, P. Edelbroek, J. van Hooff, L. Stolk, J. Pharm. Biomed. Anal. 44 (2006) 658-664; A. Allanson, M. Cotton, J. Tettey, et al., J. Pharm. Biomed. Anal. 44 (2007) 963-969] and developed a new method, namely the impregnation of sampling paper with a solution of plasma-protein, formic acid and ammonium acetate, in combination with the extraction of the blood spot by filter filtration. This kind of sample preparation provides new possibilities for blood spot sampling especially if analytes are adsorbed to the paper. The dried blood spot was analysed using the HPLC-electrospray-tandem mass spectrometry method, with 32-desmethoxyrapamycin as the internal standard. The working range of our study was 2-30 microg/l. Within this range, intra-and inter-assay variability for precision and accuracy was <15%. Everolimus blood spot samples proved stable for 3 days at 60 degrees C and for 32 days at 4 degrees C. Everolimus concentrations of one stable out-patient were compared after both blood spot sampling and conventional venous sampling on various occasions. Results indicate that this new method is promising for therapeutic drug monitoring in stable renal transplant patients.

Microanalysis of amoxicillin, flucloxacillin, and rifampicin in neonatal plasma.

Simple and rapid reversed-phase high-performance liquid chromatographic assays with ultraviolet detection have been developed and validated for the determination of amoxicillin, flucloxacillin and rifampicin in neonatal plasma. Plasma samples were either precipitated with perchloric acid (amoxicillin) or methanol (rifampicin) or extracted with methylene chloride (flucloxacillin). Precision coefficients of variation and inaccuracy were less than 15% for all three assays. Only small sample volumes (20-40 microL) were required, making the assays suitable for therapeutic drug monitoring and pharmacokinetic studies in preterm and term neonates. The assays have successfully been applied to analysis of amoxicillin, flucloxacillin and rifampicin in previously published pharmacokinetic studies in neonates.

Amoxicillin pharmacokinetics in (preterm) infants aged 10 to 52 days: effect of postnatal age.

The pharmacokinetic parameters of amoxicillin were determined in 32 newborn infants aged 10 to 52 days (mean postnatal age, 24.7 +/- 12.4 days) to improve amoxicillin dosing in this age group. Amoxicillin plasma concentrations were determined using reversed-phase high-performance liquid chromatography in surplus plasma samples from routine gentamicin assays. Amoxicillin pharmacokinetic parameters (mean +/- SD) were as follows: first-order elimination constant (K(el)) = 0.27 +/- 0.10 h(-1), volume of distribution corrected for body weight (V/W) = 0.66 +/- 0.27 L/kg, total body clearance corrected for body weight (CL/W) = 0.18 +/- 0.10 Lkg(-1)h(-1), and elimination half-life (t(1/2)) = 3.0 +/- 1.3 hours. Amoxicillin body clearance was approximately twofold greater in our patients compared with published values in younger neonates (mean postnatal age, 0.76 +/- 1.57 days). Simulation studies using the observed amoxicillin pharmacokinetic data suggest an amoxicillin dose of 40 mg/kg administered every 8 hours in infants older than 9 days postnatal age, independent of gestational age and postconceptional age, to achieve satisfactory target plasma amoxicillin concentrations less than 140 mg/L and time above minimum inhibitory concentration of at least 40%. Prospective evaluation of this suggested new dosage regimen is necessary before implementation in the care of ill neonates.

Therapeutic drug monitoring of tacrolimus with the dried blood spot method.

In a preliminary investigation an assay for tacrolimus based on fingerprick sampling and consecutive application as a blood spot on sampling paper has been developed. The dried blood spot was analysed by HPLC-tandem mass spectrometry. The validated range was 1-30 microg/l. Intra- and inter-assay variability for precision and accuracy was <7.5% and 15%, respectively. Tacrolimus concentrations of 24 stable out patients were compared after both blood spot sampling and conventional venous sampling. Method agreement was investigated with the methods of Passing and Bablok and Bland Altman and proved suitable for clinical use. The dried blood spot method for tacrolimus seems promising for patient monitoring.

Toxic rise of clozapine plasma concentrations in relation to inflammation.

Recently a small number of patients were observed in two psychiatric hospitals in the Netherlands with clozapine intoxications that complicate or mimic infections. These patients were on chronic medication and normally had stable clozapine blood plasma levels. This article presents four of these cases. Medline was searched for reports of similar cases. A hypothesis was formulated and tested by literature study. Immune modulatory and toxic effects of clozapine protein reactive metabolites or haptens, may play a role in the development of inflammation. Clozapine has a direct influence on different cytokines resembling an inflammatory reaction. Infection or inflammation could induce bioactivation of clozapine into its nitrenium ion that can exert a toxic reaction that induces apoptosis and gives rise to elevated cytokine levels. Clozapine can function as a hapten and induce an IgG, IgM or IgE mediated hypersensitivity reaction. The cytokines released during infection or inflammation downregulate the clozapine metabolism in the P450 system through CYP 1A2. Clozapine plasma levels should be monitored closely if an inflammatory or infectious process is suspected.

Comparison between human pharmacokinetics and imaging properties of two conjugation methods for 99mTc-annexin A5.

Annexin A5 (AnxA5) is a protein with high affinity for phosphatidyl serine, a phospholipid exposed on the cell surface during apoptosis. This phenomenon has been used for determination of cell death after myocardial infarction. To evaluate the potential of (99m)Tc-AnxA5 for in vivo scintigraphy of apoptotic cells, the pharmacokinetics and imaging properties of two radiopharmaceuticals, (99m)Tc-(n-1-imino-4-mercaptobutyl)-AnxA5 (I-AnxA5) and (99m)Tc-(4,5-bis(thioacetamido)pentanoyl)-AnxA5 (B-AnxA5), were studied. I-AnxA5 was administered intravenously to seven patients and one healthy volunteer, and B-AnxA5 was administered to 12 patients. All patients in the pharmacokinetic study had myocardial disease. Additionally, imaging was performed in a patient with acute myocardial infarction, as well as in three patients with different malignancies. The plasma concentration, excretion and biodistribution of (99m)Tc-AnxA5 were measured, as well as levels of AnxA5 antigen. The kinetic data of both radiopharmaceuticals in plasma fitted a two-compartment model. Both preparations had similar half-lives, but a different distribution over the two compartments. Plasma levels of AnxA5 antigen showed a broad variation. Both radiopharmaceuticals accumulated in the kidney, liver and gut. B-AnxA5 was excreted significantly faster than I-AnxA5. Both compounds can be used for imaging of the head/neck region, the thorax and the extremities. B-AnxA5 has a faster clearance and a lower radiation dose. Imaging of apoptosis in the abdomen will be difficult with both radiopharmaceuticals, and especially with B-AnxA5 because of its faster appearance in the gut.

Population pharmacokinetics and relationship between demographic and clinical variables and pharmacokinetics of gentamicin in neonates.

Population pharmacokinetic parameter estimates were calculated from 725 routine plasma gentamicin concentrations obtained in 177 neonates of 24 to 42 weeks' gestational age in their first week of life. Kel increases and V/W decreases with increasing gestational age. Almost identical results were obtained with iterative two-stage Bayesian fitting (MW\PHARM 3.30) as with a non-parametric maximization algorithm (NPEM2). The effect of various covariates on drug disposition was investigated retrospectively using multiple regression analysis. Predictive power for Kel increases with rising gestational age. For neonates 28.5 weeks and 30.9 weeks (r2 = 0.482), with gestational age, postnatal age, and Apgar score at 5 minutes being predictors. A very strong correlation existed between volume of distribution and weight (r2 = 0.83). Volume as a function of weight could be described with low predictivity by gestational age and to a lesser degree by Apgar score at 5 minutes (r2 = 0.298). The developed models need appropriate prospective clinical validation.

The relative bioavailability of metoprolol following oral and rectal administration to volunteers and patients.

The pharmacokinetics, efficacy and safety of metoprolol tartrate 25 mg fatty suppositories were studied in 5 healthy volunteers and in 8 patients suffering from instable angina pectoris. Metoprolol 25 mg capsules were used as a control oral dosage form. Metoprolol showed a considerable rectal bioavailability (AUC, C max) and was absorbed quickly from the rectum (T max). In both groups rectal bioavailability was comparable. However, oral bioavailability was much lower in the volunteer group than in the patient group. Furthermore, ratios of metoprolol/alpha-OH-metoprolol concentrations in plasma and urine gave an indication for a partial avoidance of the first pass effect after rectal administration. Further research is necessary to define an exact rectal dosage of metoprolol. In all patients, a substantial drop in heart rate, systolic and diastolic blood pressure was seen after administration of the first suppository. Metoprolol suppositories appear to be an effective, safe and suitable alternative for patients who are in need for beta blocking medication and who are unable to take oral medication for a certain amount of time.

Detection of laxative abuse by urine analysis with HPLC and diode array detection.

A simple method is proposed for analysis of stimulant laxatives and metabolites of laxatives in urine. All stimulant laxatives commercially available in Germany, Belgium and the Netherlands, the diphenylmethane derivatives and the anthraquinones, were included. Chromatography was performed with a standardized isocratic HPLC system with diode array detection ('STIP'), which is commonly used in the Netherlands for toxicological screening. The method was validated by ingestion of a normal dose of the laxatives by human volunteers. In all cases the expected laxative metabolite could be detected in urine twelve hours after intake. Also urine samples of patients, suspected of laxative abuse, were analyzed.