Cytogenetic Effects of Chronic Methylphenidate Treatment and Chronic Social Stress in Adults with Attention-Deficit/Hyperactivity Disorder.

INTRODUCTION: Methylphenidate (MPH) is widely used to treat childhood and adult attention-deficit/hyperactivity disorder (ADHD). However, there are still safety concerns about side effects in long-term treatment. The aim of this study was to assess cytogenetic effects of chronic MPH treatment in adult ADHD and to find out if chronic social stress is attenuated by medication and to investigate whether chronic psychosocial stress leads to mutagenic effects by itself. METHODS: Lymphocytes for micronucleus assay and saliva samples for cortisol measurement were collected from adult ADHD patients and healthy controls. Stress exposure of the last 3 months was assessed by TICS (Trier Inventory for Chronic Stress). RESULTS: We could not detect an influence of MPH treatment on cytogenetic markers. ADHD patients displayed significantly higher chronic stress levels measured by TICS compared to healthy controls which were influenced by duration of MPH treatment. ADHD patients also showed significantly lower basal cortisol levels. DISCUSSION: We could corroborate that there are neither cytogenetic effects of chronic stress nor of chronic MPH intake even after several years of treatment.

Gastric pH and residual volume after 1 and 2 h fasting time for clear fluids in children†.

INTRODUCTION: Current guidelines suggest a fasting time of 2 h for clear fluids, which is often exceeded in clinical practice, leading to discomfort, dehydration and stressful anaesthesia induction to patients, especially in the paediatric population. Shorter fluid fasting might be a strategy to improve patient comfort but has not been investigated yet. This prospective clinical trial compares gastric pH and residual volume after 1 vs 2 h of preoperative clear fluid fasting. METHODS: Children (1-16 yr, ASA I or II) undergoing elective procedures in general anaesthesia requiring tracheal intubation were randomized into group A with 60 min or B with 120 min preoperative clear fluid fasting. To determine gastric pH and residual volume, the gastric content was sampled in supine, left and right lateral patient position using an oro-gastric tube after intubation. Data are median (interquartile range) for group A or B (P<0.05). RESULTS: In total, 131 children aged 1.01-16.23 yr were included; gastric pH was determined in 120 cases. Patient characteristic data were similar between the two groups, except for gender (46/33 males in group A/B; P=0.02). Despite significantly shorter fasting times for clear fluids in group A compared with group B (76/136 min; P<0.001), no significant difference was observed regarding gastric pH [1.43 (1.30-1.56)/1.44 (1.29-1.68), P=0.66] or residual volume [0.43 (0.21-0.84)/0.46 (0.19-0.78) ml kg(-1), P=0.47]. CONCLUSION: One hour clear fluid fasting does not alter gastric pH or residual volume significantly compared with 2 h fasting. CLINICAL TRIAL REGISTRATION: The study was approved by the local ethics committee (KEK-ZH-Nr. 2011-0034) and registered with ClinicalTrials.gov (NCT01516775).

Genome-wide analysis of rare copy number variations reveals PARK2 as a candidate gene for attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental disorder. Genetic loci have not yet been identified by genome-wide association studies. Rare copy number variations (CNVs), such as chromosomal deletions or duplications, have been implicated in ADHD and other neurodevelopmental disorders. To identify rare (frequency ≤1%) CNVs that increase the risk of ADHD, we performed a whole-genome CNV analysis based on 489 young ADHD patients and 1285 adult population-based controls and identified one significantly associated CNV region. In tests for a global burden of large (>500 kb) rare CNVs, we observed a nonsignificant (P=0.271) 1.126-fold enriched rate of subjects carrying at least one such CNV in the group of ADHD cases. Locus-specific tests of association were used to assess if there were more rare CNVs in cases compared with controls. Detected CNVs, which were significantly enriched in the ADHD group, were validated by quantitative (q)PCR. Findings were replicated in an independent sample of 386 young patients with ADHD and 781 young population-based healthy controls. We identified rare CNVs within the parkinson protein 2 gene (PARK2) with a significantly higher prevalence in ADHD patients than in controls (P=2.8 × 10(-4) after empirical correction for genome-wide testing). In total, the PARK2 locus (chr 6: 162 659 756-162 767 019) harboured three deletions and nine duplications in the ADHD patients and two deletions and two duplications in the controls. By qPCR analysis, we validated 11 of the 12 CNVs in ADHD patients (P=1.2 × 10(-3) after empirical correction for genome-wide testing). In the replication sample, CNVs at the PARK2 locus were found in four additional ADHD patients and one additional control (P=4.3 × 10(-2)). Our results suggest that copy number variants at the PARK2 locus contribute to the genetic susceptibility of ADHD. Mutations and CNVs in PARK2 are known to be associated with Parkinson disease.

Bipolar disorder risk alleles in children with ADHD.

Bipolar disorder (BD) and attention deficit/hyperactivity disorder (ADHD) may share common genetic risk factors as indicated by the high co-morbidity of BD and ADHD, their phenotypic overlap especially in pediatric populations, the high heritability of both disorders, and the co-occurrence in families. We therefore examined whether known polygenic BD risk alleles are associated with ADHD. We chose the eight best SNPs of the recent genome-wide association study (GWAS) of BD patients of German ancestry and the nine SNPs from international GWAS meeting a 'genome-wide significance' level of α = 5 × 10(-8). A GWAS was performed in 495 ADHD children and 1,300 population-based controls using HumanHap550v3 and Human660 W-Quadv1 BeadArrays. We found no significant association of childhood ADHD with single BD risk alleles surviving adjustment for multiple testing. Yet, risk alleles for BD and ADHD were directionally consistent at eight of nine loci with the strongest support for three SNPs in or near NCAN, BRE, and LMAN2L. The polygene analysis for the BP risk alleles at all 14 loci indicated a higher probability of being a BD risk allele carrier in the ADHD cases as compared to the controls. At a moderate power to detect association with ADHD, if true effects were close to estimates from GWAS for BD, our results suggest that the possible contribution of BD risk variants to childhood ADHD risk is considerably lower than for BD. Yet, our findings should encourage researchers to search for common genetic risk factors in BD and childhood ADHD in future studies.

A cooperative interaction between LPHN3 and 11q doubles the risk for ADHD.

In previous studies of a genetic isolate, we identified significant linkage of attention deficit hyperactivity disorder (ADHD) to 4q, 5q, 8q, 11q and 17p. The existence of unique large size families linked to multiple regions, and the fact that these families came from an isolated population, we hypothesized that two-locus interaction contributions to ADHD were plausible. Several analytical models converged to show significant interaction between 4q and 11q (P<1 × 10(-8)) and 11q and 17p (P<1 × 10(-6)). As we have identified that common variants of the LPHN3 gene were responsible for the 4q linkage signal, we focused on 4q-11q interaction to determine that single-nucleotide polymorphisms (SNPs) harbored in the LPHN3 gene interact with SNPs spanning the 11q region that contains DRD2 and NCAM1 genes, to double the risk of developing ADHD. This interaction not only explains genetic effects much better than taking each of these loci effects by separated but also differences in brain metabolism as depicted by proton magnetic resonance spectroscopy data and pharmacogenetic response to stimulant medication. These findings not only add information about how high order genetic interactions might be implicated in conferring susceptibility to develop ADHD but also show that future studies of the effects of genetic interactions on ADHD clinical information will help to shape predictive models of individual outcome.

Form follows function: morphological diversification and alternative trapping strategies in carnivorous Nepenthes pitcher plants.

Carnivorous plants of the genus Nepenthes have evolved a striking diversity of pitcher traps that rely on specialized slippery surfaces for prey capture. With a comparative study of trap morphology, we show that Nepenthes pitcher plants have evolved specific adaptations for the use of either one of two distinct trapping mechanisms: slippery wax crystals on the inner pitcher wall and 'insect aquaplaning' on the wet upper rim (peristome). Species without wax crystals had wider peristomes with a longer inward slope. Ancestral state reconstructions identified wax crystal layers and narrow, symmetrical peristomes as ancestral, indicating that wax crystals have been reduced or lost multiple times independently. Our results complement recent reports of nutrient source specializations in Nepenthes and suggest that these specializations may have driven speciation and rapid diversification in this genus.

No evidence for association between a functional promoter variant of the Norepinephrine Transporter gene SLC6A2 and ADHD in a family-based sample.

Noradrenergic neurotransmission influences executive functions, attentional performance, and general alertness, involving neuronal networks affected in attention deficit/hyperactivity disorder (ADHD). The norepinephrine transporter facilitates the reuptake of norepinephrine and dopamine in the prefrontal cortex and represents the main target of atomoxetine, an effective drug in the treatment of ADHD. Due to its influence on catecholaminergic signaling, variants of the coding gene (SLC6A2) have been widely investigated in ADHD. Several previous studies report an association between single nucleotide polymorphisms located in SLC6A2 and ADHD; however, the findings are inconsistent. The variant A-3081T (rs28386840) has been shown to have major influence on the expression levels of SLC6A2 due to sequence alteration at a repressor binding site, with the T-allele being associated with ADHD. We tested this potential association of A-3081T in a German family-based ADHD sample of 235 children from 162 families, which has a power >99% based on the previously reported odds ratios. There was no evidence for an overtransmission of the risk allele T (transmission rate: 48.5%, P = 0.55). We conclude that A-3081T is not a major risk variant in our ADHD sample, though SLC6A2 remains an interesting candidate gene in ADHD, especially for the inattentive subtype.

Genome-wide copy number variation analysis in attention-deficit/hyperactivity disorder: association with neuropeptide Y gene dosage in an extended pedigree.

Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental syndrome characterized by hyperactivity, inattention and increased impulsivity. To detect micro-deletions and micro-duplications that may have a role in the pathogenesis of ADHD, we carried out a genome-wide screen for copy number variations (CNVs) in a cohort of 99 children and adolescents with severe ADHD. Using high-resolution array comparative genomic hybridization (aCGH), a total of 17 potentially syndrome-associated CNVs were identified. The aberrations comprise 4 deletions and 13 duplications with approximate sizes ranging from 110 kb to 3 Mb. Two CNVs occurred de novo and nine were inherited from a parent with ADHD, whereas five are transmitted by an unaffected parent. Candidates include genes expressing acetylcholine-metabolizing butyrylcholinesterase (BCHE), contained in a de novo chromosome 3q26.1 deletion, and a brain-specific pleckstrin homology domain-containing protein (PLEKHB1), with an established function in primary sensory neurons, in two siblings carrying a 11q13.4 duplication inherited from their affected mother. Other genes potentially influencing ADHD-related psychopathology and involved in aberrations inherited from affected parents are the genes for the mitochondrial NADH dehydrogenase 1 α subcomplex assembly factor 2 (NDUFAF2), the brain-specific phosphodiesterase 4D isoform 6 (PDE4D6) and the neuronal glucose transporter 3 (SLC2A3). The gene encoding neuropeptide Y (NPY) was included in a ∼3 Mb duplication on chromosome 7p15.2-15.3, and investigation of additional family members showed a nominally significant association of this 7p15 duplication with increased NPY plasma concentrations (empirical family-based association test, P=0.023). Lower activation of the left ventral striatum and left posterior insula during anticipation of large rewards or losses elicited by functional magnetic resonance imaging links gene dose-dependent increases in NPY to reward and emotion processing in duplication carriers. These findings implicate CNVs of behaviour-related genes in the pathogenesis of ADHD and are consistent with the notion that both frequent and rare variants influence the development of this common multifactorial syndrome.

A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication.

Attention-Deficit/Hyperactivity Disorder (ADHD) has a very high heritability (0.8), suggesting that about 80% of phenotypic variance is due to genetic factors. We used the integration of statistical and functional approaches to discover a novel gene that contributes to ADHD. For our statistical approach, we started with a linkage study based on large multigenerational families in a population isolate, followed by fine mapping of targeted regions using a family-based design. Family- and population-based association studies in five samples from disparate regions of the world were used for replication. Brain imaging studies were performed to evaluate gene function. The linkage study discovered a genome region harbored in the Latrophilin 3 gene (LPHN3). In the world-wide samples (total n=6360, with 2627 ADHD cases and 2531 controls) statistical association of LPHN3 and ADHD was confirmed. Functional studies revealed that LPHN3 variants are expressed in key brain regions related to attention and activity, affect metabolism in neural circuits implicated in ADHD, and are associated with response to stimulant medication. Linkage and replicated association of ADHD with a novel non-candidate gene (LPHN3) provide new insights into the genetics, neurobiology, and treatment of ADHD.

Exploring the genetic link between RLS and ADHD.

Attention deficit/hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder of childhood onset. Clinical and biological evidence points to shared common central nervous system (CNS) pathology of ADHD and restless legs syndrome (RLS). It was hypothesized that variants previously found to be associated with RLS in two large genome-wide association studies (GWA), will also be associated with ADHD. SNPs located in MEIS1 (rs2300478), BTBD9 (rs9296249, rs3923809, rs6923737), and MAP2K5 (rs12593813, rs4489954) as well as three SNPs tagging the identified haplotype in MEIS1 (rs6710341, rs12469063, rs4544423) were genotyped in a well characterized German sample of 224 families comprising one or more affected sibs (386 children) and both parents. We found no evidence for preferential transmission of the hypothesized variants to ADHD. Subsequent analyses elicited nominal significant association with haplotypes consisting of the three SNPs in BTBD9 (chi2 = 14.8, df = 7, nominal p = 0.039). According to exploratory post hoc analyses, the major contribution to this finding came from the A-A-A-haplotype with a haplotype-wise nominal p-value of 0.009. However, this result did not withstand correction for multiple testing. In view of our results, RLS risk alleles may have a lower effect on ADHD than on RLS or may not be involved in ADHD. The negative findings may additionally result from genetic heterogeneity of ADHD, i.e. risk alleles for RLS may only be relevant for certain subtypes of ADHD. Genes relevant to RLS remain interesting candidates for ADHD; particularly BTBD9 needs further study, as it has been related to iron storage, a potential pathophysiological link between RLS and certain subtypes of ADHD.

Reflective and impulsive reactions in ADHD subtypes.

Research on different subtypes of attention deficit hyperactivity disorder (ADHD) yielded inconsistent results regarding the nature of cognitive deficits. Whereas some studies report significant differences between subtypes, others fail to report these differences. In fact, the majority of studies in the field of ADHD does not differentiate between subtypes at all. The present study adopted the cognitive task of negating valence to compare the DSM-defined ADHD combined subtype (ADHD-C; n = 25), the inattentive subtype (ADHD-I; n = 25) and the control group (n = 30). As a main result, children with ADHD-C showed significant impairments compared to children with ADHD-I and the control group on conscious responses-driven by executive function, as well as on unconscious associative tasks. Medical treatment with stimulants positively influenced cognitive performance, although to a different extent for subgroups. The results are discussed in the context of current theories of ADHD and imply indications for further research in this field.

[Neurobiology of attention-deficit hyperactivity disorder]. / Neurobiologie des Aufmerksamkeitsdefizit-/Hyperaktivitätssyndroms.

Attention-deficit hyperactivity disorder (ADHD) is defined as a clinically heterogeneous neurodevelopmental syndrome with the contribution of numerous genetic and environmental risk factors. The goal of interdisciplinary and translational neurobiological research is to clarify the interdependent relationship between molecular mechanisms and structural-functional substrates in the pathogenesis of ADHD and its significance to the disorder's long-term course. Work on ADHD-specific molecular genetic and developmental biological essentials of brain function and on the structural-functional basis of behavior holds the promise of developing predictors and differential strategies for effective therapy of severe and chronic courses of ADHD. To define evolutionary conserved ADHD-relevant principles of structure and function of the brain and behavior typical to the syndrome, an integrated approach in the elucidation of specific neuro- and psychobiological mechanisms and thus systemic pathophysiology of ADHD is crucial. Regarding compromised neurodevelopment, pathophysiological models of ADHD, particularly its syndromal and comorbid dimensions, therefore require the combination of molecular genetic, neuroimaging, neuropsychology, behavioral, and psychosocial strategies to explain complete causal chains.

Allelic variants of SNAP25 in a family-based sample of ADHD.

Altered neurotransmission has been suggested to be a crucial factor in the pathophysiology of attention-deficit/hyperactivity disorder ADHD. Subsequently genes encoding for synaptic proteins have been investigated in candidate gene studies. These proteins mediate the release of neurotransmitters into the synaptic cleft in the process of signal transduction by forming a transient complex, enabling the junction of vesicle and synaptic membrane. One of the core proteins of this complex is the synaptosomal-associated protein 25 (SNAP25). It is one of the most validated candidate genes in ADHD according to meta-analyses. However, differing results were observed in previous studies, some of which were not able to observe association with ADHD. In this study we aimed to investigate association of genetic variants of SNAP25 located in the putative promoter region of SNAP25 and a SNP in intron 8, previously reported to associated with ADHD. A family based design was applied to detect preferential transmission of genetic variants. In our German ADHD sample no preferential transmission of either variant could be observed. Further investigation considering sub-sample analysis regarding response to D-amphetamine could enlight the role of SNAP25 in ADHD.

Genome-wide linkage analysis of ADHD using high-density SNP arrays: novel loci at 5q13.1 and 14q12.

Previous genome-wide linkage studies applied the affected sib-pair design; one investigated extended pedigrees of a genetic isolate. Here, results of a genome-wide high-density linkage scan of attention-deficit/hyperactivity disorder (ADHD) using an array-based genotyping of approximately 50 K single nucleotide polymorphism (SNPs) markers are presented. We investigated eight extended pedigrees of German origin that were non-related, not part of a genetic isolate and ascertained on the basis of clinical referral. Two parametric analyses maximizing LOD scores (MOD) and a non-parametric analysis for both a broad and a narrow phenotype approach were conducted. Novel linkage loci across all families were detected at 2q35, 5q13.1, 6q22-23 and 14q12, within individual families at 18q11.2-12.3. Further linkage regions at 7q21.11, 9q22 and 16q24.1 in all families, and at 1q25.1, 1q25.3, 9q31.1-33.1, 9q33, 12p13.33, 15q11.2-13.3 and 16p12.3-12.2 in individual families replicate previous findings. High-resolution linkage mapping points to several novel candidate genes characterized by dense expression in the brain and potential impact on disorder-relevant synaptic transmission. Our study provides further evidence for common gene effects throughout different populations despite the complex multifactorial etiology of ADHD.

Association and linkage of allelic variants of the dopamine transporter gene in ADHD.

Previously, we had reported a genome-wide scan for attention-deficit/hyperactivity disorder (ADHD) in 102 families with affected sibs of German ancestry; the highest multipoint LOD score of 4.75 was obtained on chromosome 5p13 (parametric HLOD analysis under a dominant model) near the dopamine transporter gene (DAT1). We genotyped 30 single nucleotide polymorphisms (SNPs) in this candidate gene and its 5' region in 329 families (including the 102 initial families) with 523 affected offspring. We found that (1) SNP rs463379 was significantly associated with ADHD upon correction for multiple testing (P=0.0046); (2) the global P-value for association of haplotypes was significant for block two upon correction for all (n=3) tested blocks (P=0.0048); (3) within block two we detected a nominal P=0.000034 for one specific marker combination. This CGC haplotype showed relative risks of 1.95 and 2.43 for heterozygous and homozygous carriers, respectively; and (4) finally, our linkage data and the genotype-IBD sharing test (GIST) suggest that genetic variation at the DAT1 locus explains our linkage peak and that rs463379 (P<0.05) is the only SNP of the above haplotype that contributed to the linkage signal. In sum, we have accumulated evidence that genetic variation at the DAT1 locus underlies our ADHD linkage peak on chromosome 5; additionally solid association for a single SNP and a haplotype were shown. Future studies are required to assess if variation at this locus also explains other positive linkage results obtained for chromosome 5p.

No evidence for preferential transmission of common valine allele of the Val66Met polymorphism of the brain-derived neurotrophic factor gene (BDNF) in ADHD.

Attention deficit/hyperactivity disorder (ADHD) is a highly heritable common neurodevelopmental disorder with onset in childhood. A coding SNP (rs6265, Val66Met) of the brain-derived neurotrophic factor gene (BDNF) has recently been associated with ADHD. More specifically, paternal over-transmission of the common Val66 allele to affected children had been observed. We aimed to confirm these findings in a large, sufficiently powered, and well characterized German ADHD family sample. The Val66Met polymorphism of BDNF was genotyped in 294 families comprising one or more affected sibs (468 children). Contrary to previous reports, we did not observe over-transmission of the common Val66 allele, from either parent to affected children. We did not find support for an involvement of the Val66 allele of the Val66Met polymorphism of BDNF in the pathogenesis of ADHD in our sample.

Transmission disequilibrium of polymorphic variants in the tryptophan hydroxylase-2 gene in attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) is the most common behavioral disorder in childhood with substantial heritability. Pharmacological and molecular genetic studies as well as characterization of animal models have implicated serotonergic dysfunction in the pathophysiology of ADHD. Here, we investigated the effect of polymorphic variants in the gene of the tryptophan hydroxylase-2 (TPH2), the rate-limiting enzyme of serotonin (5-HT) synthesis in the brain, in children and adolescents with ADHD. We analyzed three single nucleotide polymorphisms (SNPs) in and downstream of the transcriptional control region of the TPH2 gene in 103 families with 225 affected children. Allelic association in families with more than one affected child was assessed using the pedigree disequilibrium test. Preferential transmissions were detected for the two SNPs in TPH2's regulatory region (rs4570625, P=0.049; rs11178997, P=0.034), but not for the third SNP in intron 2 (rs4565946, P=0.3517). Haplotype analysis revealed a strong trend of association between the regulatory region SNPs (rs4570625, rs11178997) and ADHD (P=0.064). Our results link potentially functional TPH2 variations to the pathophysiology of ADHD, and further support the relevance of 5-HT in disorders related to altered motor activity and cognitive processes.

Biomonitoring of exposure to polycyclic aromatic hydrocarbons of nonoccupationally exposed persons.

In a field study with 69 subjects, we investigated the influence of smoking, exposure to environmental tobacco smoke (ETS), diet, and location of residence on biomarkers for polycyclic aromatic hydrocarbons (PAH), including urinary excretion of 1-hydroxypyrene and benzo[a]pyrene (BaP) adducts of hemoglobin and albumin. The self-reported smoking status and the extent of ETS exposure were verified by urinary cotinine measurements. ETS exposure was quantified by nicotine and 3-ethenylpyridine measurements on personal samplers worn by the nonsmokers over 5 or 7 days before blood and urine samples were collected. Smokers (n = 27), on average, excreted 0.346 microg/24 h 1-hydroxypyrene, whereas the corresponding value for nonsmokers (n = 42) was 0.157 microg/24 h. Average BaP adduct levels with hemoglobin and albumin were 0.105 fmol/mg and 0.042 fmol/mg, respectively, for smokers, and 0.068 fmol/mg and 0.020 fmol/mg, respectively, for nonsmokers. The differences, except for the hemoglobin adducts, were statistically significant. Of the 42 nonsmokers, 19 were classified as passive smokers. There was no significant difference in the PAH biomarkers between nonsmokers exposed to ETS and those not or rarely exposed to ETS. Total dietary BaP intake, as calculated from questionnaire data, did not correlate with any of the PAH biomarkers (r < 0.1). Subjects living in the suburbs tended to have higher BaP-protein adduct levels than subjects living in the city. Our findings suggest that diet and smoking are major sources for PAH exposure of persons not occupationally exposed to PAH, whereas the influence of ETS exposure is negligible. The lack of correlation between the dietary PAH intake and the PAH biomarkers may be due to the inaccuracy of the estimate for the dietary PAH intake.

Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2'-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection.

Exogenous and endogenous oxidants constantly cause oxidative damage to DNA. Since the reactive oxidants itself are not suitable for analysis, oxidized bases like 8-hydroxy-2'-deoxyguanosine (8OHdG) are used as biomarkers for oxidative stress, either in cellular DNA or as elimination product in urine. A simple, fast and robust analytical procedure is described for urinary 8OHdG as an indicator of oxidative damage in humans. The adduct was purified from human urine by applying a single solid-phase extraction step on LiChrolut EN. After evaporation of the eluate, the residue was resolved and an aliquote was injected into a HPLC system with a triple quadrupole mass spectrometer. The limit of detection was 0.2 ng ml(-1) (7 fmol absolute) when using one product ion as quantifier and two further product ions as qualifier. The coefficient of variation was 10.1% (n=5 at 2.8 ng ml(-1) urine). The sample throughput was about 50 samples a day. Thus, this method is more sensitive and much faster than the common method using HPLC with electrochemical detection. The results of a study with nine volunteers investigated at six time-points each over 5 days are presented. The mean excretion of 8OHdG was 2.1 ng mg(-1) creatinine (range 0.17-5.9 ng mg(-1) creatinine; 4 of 53 samples were below the LOD). A relatively large intra- (relative SD 66%) and inter-individual (relative SD 71%) variation in urinary 8OHdG excretion rates was found.

Determination of sorbic acid in urine by gas chromatography-mass spectrometry.

The average daily uptake of the common food preservative sorbic acid is estimated to range from 0.01 to 1.1 mg kg-1. Sorbic acid mainly is metabolised to carbon dioxide. Minor amounts are converted to trans,trans-muconic acid (ttMA) as well as excreted unchanged into the urine. Since urinary ttMA is a biomarker for the occupational and environmental exposure to benzene, there is an additional need for monitoring the uptake of sorbic acid, particularly at low, environmental benzene exposure levels. For this purpose, a simple, robust and rapid method for the determination of sorbic acid in urine at trace levels was developed. After addition of 10 ml of water and 5 ml of 8 M hydrochloric acid to 10 ml of the thawed urine, the sample was water steam distilled using an automated distillation device. A total of 100 ml of the distillate were solid-phase extracted. After washing, the sorbic acid was eluted with 4 ml methanol. The eluate was reduced under a stream of nitrogen to a volume of 300 microliters. After addition of 500 microliters boron trifluoride in methanol and incubation for 1 h at 60 degrees C, the resulting sorbic acid methyl ester was extracted three times with 1 ml heptane. To the combined heptane layers, sorbic acid ethyl ester was added as an internal standard. After reducing to a volume of 100 microliters in a stream of nitrogen, the final analysis was performed by GC-MS using the fragment ions m/z 126 for the analyte and m/z 140 for the internal standard. The limit of detection was 0.7 ng ml-1 urine and the R.S.D. of 69 duplicate determinations was 7.5%. In a controlled, experimental study and in a field study, we were able to show that urinary sorbic acid is a marker for the dietary uptake of sorbic acid and that sorbic acid is converted to ttMA. On average, 0.1% of the dietary sorbic acid is excreted unchanged into the urine. Excretion is complete within 24 h. We found that, on average, 0.23% of the oral dose of sorbic acid is excreted as urinary ttMA. There was a significant correlation between urinary excretions of sorbic acid and ttMA (r = 0.74, n = 69). We conclude that urinary sorbic acid can be used to correct the urinary ttMA level in order to determine the portion related to benzene exposure. This appears to be necessary particularly at low, environmental benzene levels.