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PubMed | Ludwig Maximilians University of Munich, Institute of Biomedical Problems, University of The Sunshine Coast, University of Burgundy and Institute of Doping Analysis and Sports Biochemistry
Type: | Journal: Progress in neuro-psychopharmacology & biological psychiatry | Year: 2016

Increasing evidence indicates that chronic stress, such as social isolation, plays an important role in the development of a variety of psychiatric and somatic disorders. Meanwhile, chronic stress imposed by prolonged isolation and confinement in the spacecraft is also one of the major concerns for the health of future interplanetary space travelers. Preclinical studies suggest that the peripheral endocannabinoid (eCB) system is involved in the regulation of the stress response and eCB signaling is implicated in the pathogenesis of stress-related diseases. However, there are only few human studies addressing this topic, of which most focusing on patients who have already developed a certain type of disorder. It remains unknown whether chronic stress may affect eCB signaling in healthy humans. A 520-d isolation and confinement study simulating a flight to Mars provided an extraordinary chance to study the effects of prolonged stress in healthy humans. During the study period, the participants lived in confinement and could not meet their families, friends, or strangers for more than 500 days. We examined the impact of chronic exposure to isolation and confinement through monitoring their psychological state, brain cortical activity, sympathetic adrenal-medullary system response and eCB signaling response. We observed reduced positive emotion ratings, decreased brain cortical activities and high levels of catecholamine release, indicating that prolonged exposure to isolation and confinement stressors may bring about changes both psychologically and physiologically. Importantly, for eCB signaling response, blood concentrations of eCB 2-arachidonoylglycerol (2-AG), but not anandamide (AEA), were significantly reduced (p<0.001), suggesting that dysregulation of 2-AG signaling might be specifically implicated in the response to chronic stressors.


Auwarter V.,Albert Ludwigs University of Freiburg | Wohlfarth A.,Albert Ludwigs University of Freiburg | Traber J.,Albert Ludwigs University of Freiburg | Thieme D.,Institute of Doping Analysis and Sports Biochemistry | Weinmann W.,Albert Ludwigs University of Freiburg
Forensic Science International | Year: 2010

Differentiation between external contamination and incorporation of drugs or their metabolites from inside the body via blood, sweat or sebum is a general issue in hair analysis and of high concern when interpreting analytical results. In hair analysis for cannabinoids the most common target is Δ9-tetrahydrocannabinol (THC), sometimes cannabidiol (CBD) and cannabinol (CBN) are determined additionally. After repeated external contamination by cannabis smoke these analytes are known to be found in hair even after performing multiple washing steps. A widely accepted strategy to unequivocally prove active cannabis consumption is the analysis of hair extracts for the oxidative metabolite 11-nor-9-carboxy-THC (THC-COOH). Although the acidic nature of this metabolite suggests a lower rate of incorporation into the hair matrix compared to THC, it is not fully understood up to now why hair concentrations of THC-COOH are generally found to be much lower (mostly <10 pg/mg) than the corresponding THC concentrations. Δ9-Tetrahydrocannabinolic acid A (THCA A) is the preliminary end product of the THC biosynthesis in the cannabis plant. Unlike THC it is non-psychoactive and can be regarded as a 'precursor' of THC being largely decarboxylated when heated or smoked. The presented work shows for the first time that THCA A is not only detectable in blood and urine of cannabis consumers but also in THC positive hair samples. A pilot experiment performed within this study showed that after oral intake of THCA A on a regular basis no relevant incorporation into hair occurred. It can be concluded that THCA A in hair almost exclusively derives from external contamination e.g. by side stream smoke. Elevated temperatures during the analytical procedure, particularly under alkaline conditions, can lead to decarboxylation of THCA A and accordingly increase THC concentrations in hair. Additionally, it has to be kept in mind that in hair samples tested positive for THCA A at least a part of the 'non-artefact' THC probably derives from external contamination as well, because in condensate of cannabis smoke both THC and THCA A are present in relevant amounts. External contamination by side stream smoke could therefore explain the great differences in THC and THC-COOH hair concentrations commonly found in cannabis users. © 2010 Elsevier Ireland Ltd. All rights reserved.


Chouker A.,Ludwig Maximilians University of Munich | Kaufmann I.,Ludwig Maximilians University of Munich | Kreth S.,Ludwig Maximilians University of Munich | Hauer D.,Ludwig Maximilians University of Munich | And 5 more authors.
PLoS ONE | Year: 2010

Background: A substantial number of individuals are at risk for the development of motion sickness induced nausea and vomiting (N&V) during road, air or sea travel. Motion sickness can be extremely stressful but the neurobiologic mechanisms leading to motion sickness are not clear. The endocannabinoid system (ECS) represents an important neuromodulator of stress and N&V. Inhibitory effects of the ECS on N&V are mediated by endocannabinoid-receptor activation. Methodology/Principal Findings: We studied the activity of the ECS in human volunteers (n = 21) during parabolic flight maneuvers (PFs). During PFs, microgravity conditions (<10-2 g) are generated for approximately 22 s which results in a profound kinetic stimulus. Blood endocannabinoids (anandamide and 2-arachidonoylglycerol, 2-AG) were measured from blood samples taken in-flight before start of the parabolic maneuvers, after 10, 20, and 30 parabolas, in-flight after termination of PFs and 24 h later. Volunteers who developed acute motion sickness (n = 7) showed significantly higher stress scores but lower endocannabinoid levels during PFs. After 20 parabolas, blood anandamide levels had dropped significantly in volunteers with motion sickness (from 0.39±0.40 to 0.22±0.25 ng/ml) but increased in participants without the condition (from 0.43±0.23 to 0.60±0.38 ng/ml) resulting in significantly higher anandamide levels in participants without motion sickness (p = 0.02). 2-AG levels in individuals with motion sickness were low and almost unchanged throughout the experiment but showed a robust increase in participants without motion sickness. Cannabinoid-receptor 1 (CB1) but not cannabinoid-receptor 2 (CB2) mRNA expression in leucocytes 4 h after the experiment was significantly lower in volunteers with motion sickness than in participants without N&V. Conclusions/Significance: These findings demonstrate that stress and motion sickness in humans are associated with impaired endocannabinoid activity. Enhancing ECS signaling may represent an alternative therapeutic strategy for motion sickness in individuals who do not respond to currently available treatments. © 2010 Choukèr et al.


Schonfelder M.,TU Munich | Hofmann H.,TU Munich | Anielski P.,Institute of Doping Analysis and Sports Biochemistry | Thieme D.,Institute of Doping Analysis and Sports Biochemistry | And 2 more authors.
Drug Testing and Analysis | Year: 2011

Doping with anabolic agents is regulated within a number of sports. Testosterone and its functional analogs are popular compounds for increasing muscle mass, physical performance, recovery, and reducing body fat. While routine tests for anabolic drugs exist (e.g. hair, urine, and blood analysis), the aim of the present study is to determine specific gene expression profiles (induced by testosterone and exercise) which may be used as effective biomarkers to determine the use of anabolic drugs. In this study, whole blood samples of 19 male volunteers were analyzed by semi-quantitative real-time polymerase chain reaction (RT-PCR) for gene expression profiles in the context of exercise and transdermal testosterone application (1.5mg/kg body weight). The hormone application was monitored by urine and saliva analysis for testosterone. Both urinary and saliva levels indicate that transdermal testosterone application leads to an increase of testosterone, especially after exercise. RT-PCR results showed a clear variation in the expression of target genes as well as established housekeeping genes. Only one of the nine common housekeeping genes, cyclophilin b (PPIB), appears to be independent of both exercise and testosterone. Out of 14 candidate genes, five are unregulated; all others were more or less influenced by the mentioned variables. Only interleukin-6 appeared to be exclusively dependent on long-term testosterone application. This study indicates that many genes are not influenced by testosterone alone while exercise modulates gene expression in whole blood samples. As such, exercise must be considered when validating gene expression techniques for doping analysis. © 2011 John Wiley & Sons, Ltd.


Diepenbruck I.,University of Hamburg | Much C.C.,University of Hamburg | Krumbholz A.,Institute of Doping Analysis and Sports Biochemistry | Kolster M.,University of Hamburg | And 6 more authors.
Journal of Molecular Medicine | Year: 2013

Prenatal steroids have an undisputed positive effect of decreasing neonatal morbidity and mortality by improving fetal lung maturation. Some concerns have been raised on long-term consequences on the hypothalamic-pituitary-adrenal axis and cognition, but there are no studies addressing effects on the immune system. The thymus is an essential organ for the development and selection of T cells, and thymocytes are extremely sensitive to steroids. Using a mouse model for prenatal steroid administration, we show here that betamethasone treatment to the mother has a profound effect on the thymus of the offspring. We find the thymus volume reduced, affecting mostly the developing CD4+ CD8+ double-positive thymocytes and a compensatory accelerated transition of the earlier stages to replenish the depleted compartment. This effect lasts for at least 3 days, which correspond to a very relevant period for the selection of the T cell repertoire. Moreover, we show that low doses of betamethasone have similar effects on human thymocytes in vitro. Therefore, further studies are needed to analyze possible long-term consequences of this treatment on the immune system of the offspring. Key message: Betamethasone administered to the mother before birth reaches the fetal thymus. Prenatal betamethasone results in massive loss of developing thymocytes. The effects of betamethasone on thymus development are visible for several days. Human thymocytes are also sensitive to low doses of betamethasone. Altered thymocyte development around birth may have an effect on the immune system. © 2013 Springer-Verlag Berlin Heidelberg.


Krumbholz A.,Institute of Doping Analysis and Sports Biochemistry | Anielski P.,Institute of Doping Analysis and Sports Biochemistry | Reisch N.,Ludwig Maximilians University of Munich | Schelling G.,Ludwig Maximilians University of Munich | Thieme D.,Institute of Doping Analysis and Sports Biochemistry
Therapeutic Drug Monitoring | Year: 2013

BACKGROUND:: Endogenous corticosteroids and endocannabinoids are both known to be involved in stress adaption and anti-inflammatory and immuneregulatory effects. The application of hair as retrospective specimen for long-term recording of corticosteroids and its association with stress-induced biochemical alterations was intensively examined. METHODS:: To evaluate the stability and correlation of various parameters of the endocannabinoid and corticosteroid systems, a prospective study was carried out. Hair samples were collected monthly over a pregnancy cycle (sixth week of pregnancy to 9 weeks postpartum). By comparison of hair concentrations in particular segments (ie, grown in the same time span but collected at different times), an examination of analyte stability in hair was achieved. Additionally, the comparison of proximal segments provided on biochemical information that is independent of alteration due to physical instability. The detection limits of a validated procedure using solid-phase extraction cleanup and liquid chromatography-mass spectrometry proved to be suitable to identify the endogenous levels of cortisol (limits of detection = 1.6 pg/mg), cortisone (2.1 pg/mg), anandamide (AEA, 0.3 pg/mg), and 2-arachidonoylglycerol (15 pg/mg). RESULTS:: Corticosteroid concentrations in corresponding hair segments were found to be reduced with increasing hair age; an average decline of cortisol and cortisone by 50% in 4 months was estimated. Independently, an increase of cortisol and cortisone in proximal segments collected during pregnancy was confirmed, which is assumed to be stress related. Endocannabinoids proved to be by far more stable, as demonstrated by subsequent monthly collection of corresponding segments and there was hardly any washout of AEA detectable. Elevated hair concentrations of AEA and 2- arachidonoylglycerol were detected in the first-second trimester of pregnancy, which corresponds to negative correlations between AEA, cortisol, and cortisone. Copyright © 2013 by Lippincott Williams and Wilkins.


PubMed | Institute of Doping Analysis and Sports Biochemistry
Type: Journal Article | Journal: Drug testing and analysis | Year: 2010

The potential influence of alcohol consumption on endogenous steroids has already been described in the literature. In those studies the ethanol level after ingestion was monitored using its concentration in blood, urine or saliva. Corresponding methods are not commonly used in anti-doping laboratories. Ethylglucuronide (EtG), which can be easily detected by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), appears to be a more suitable parameter for this purpose. It is slowly excreted into the urine and indicates alcohol intake for a much longer period than blood or urinary alcohol and it is therefore routinely used for legal purposes as an alcohol consumption marker. In pharmacokinetic studies that aimed to establish calculation models after ethanol intake, the formation of EtG was observed to coincide with elevated urinary testosterone/epitestosterone (T/E) ratios. Similarly, large amounts of EtG were correlated with abnormal steroid profiles found in routine doping samples. In this pilot study, several cases with significantly elevated T/E ratios were associated with urinary EtG concentrations higher than 50 microg/mL. These findings confirmed recent intake of ethanol in considerable amounts and suggest a connection to changes in specific steroid profile parameters. Owing to the ease with which procedures to determine EtG can be carried out, and the potential for such procedures to be introduced into screening schemes, the inclusion of this marker in the final evaluation of suspicious outliers in T/E ratio longitudinal studies would seem to be very useful.


Anielski P.,Institute of Doping Analysis and Sports Biochemistry | Simmchen J.,Institute of Doping Analysis and Sports Biochemistry | Wassill L.,AmplexDiagnostics GmbH | Ganghofner D.,AmplexDiagnostics GmbH | Thieme D.,Institute of Doping Analysis and Sports Biochemistry
Drug Testing and Analysis | Year: 2011

The deletion polymorphism of the enzyme UGT2B17 is known to correlate with the level of the testosterone to epitestosterone (T/E) ratio in urine specimen. Due to the importance of the T/E ratio to detect testosterone abuse in doping analysis, a PCR-ELISA system (Genotype® UGT test, AmplexDiagnostics) was established to identify the UGT2B17 phenotype in urine samples. Epidemiological investigations in a set of 674 routine doping controls (in- and out-of-competition) resulted in 22.8% homozygote gene-deleted and 74.5% UGT2B17-positive athletes. The validated test system has shown to be robust and sensitive: in only 18 cases (2.7%) isolation of cell material from urine failed. Following hydrolysis of glucuronidated conjugates, steroids were analyzed as bis-TMS derivatives by gas chromatography-mass spectrometry (GC-MS), for example, testosterone (T) and epitestosterone (E). Additionally, isotope ration mass spectrometry (IRMS) analysis and luteinizing hormone (LH) measurement were applied. Mean T/E ratios significantly correlated with the UGT2B17 phenotype (del: T/E 0.9; pos: 1.7), however the values did not differ as distinctive as reported in previous studies. Additionally, the T/E ratios in the gene-deleted group did not show a normal curve of distribution (median of T/E 0.5). Obviously, beside the UGT2B17 deletion further influences have to be taken into account, for example, polymorphisms or induction of other metabolizing enzymes. Our results indicate that the UGT2B17 polymorphism might be insufficient when utilized solely as a crucial parameter for individual interpretation of T/E in urine. Nevertheless, the detection of the UGT2B17-gene deletion in urine samples would provide additional information important for gathering evidence in analysis of steroids in doping control. © 2011 John Wiley & Sons, Ltd.


PubMed | Institute of Doping Analysis and Sports Biochemistry
Type: Evaluation Studies | Journal: Drug testing and analysis | Year: 2011

The deletion polymorphism of the enzyme UGT2B17 is known to correlate with the level of the testosterone to epitestosterone (T/E) ratio in urine specimen. Due to the importance of the T/E ratio to detect testosterone abuse in doping analysis, a PCR-ELISA system (Genotype UGT test, AmplexDiagnostics) was established to identify the UGT2B17 phenotype in urine samples. Epidemiological investigations in a set of 674 routine doping controls (in- and out-of-competition) resulted in 22.8% homozygote gene-deleted and 74.5% UGT2B17-positive athletes. The validated test system has shown to be robust and sensitive: in only 18 cases (2.7%) isolation of cell material from urine failed. Following hydrolysis of glucuronidated conjugates, steroids were analyzed as bis-TMS derivatives by gas chromatography-mass spectrometry (GC-MS), for example, testosterone (T) and epitestosterone (E). Additionally, isotope ration mass spectrometry (IRMS) analysis and luteinizing hormone (LH) measurement were applied. Mean T/E ratios significantly correlated with the UGT2B17 phenotype (del: T/E 0.9; pos: 1.7), however the values did not differ as distinctive as reported in previous studies. Additionally, the T/E ratios in the gene-deleted group did not show a normal curve of distribution (median of T/E 0.5). Obviously, beside the UGT2B17 deletion further influences have to be taken into account, for example, polymorphisms or induction of other metabolizing enzymes. Our results indicate that the UGT2B17 polymorphism might be insufficient when utilized solely as a crucial parameter for individual interpretation of T/E in urine. Nevertheless, the detection of the UGT2B17-gene deletion in urine samples would provide additional information important for gathering evidence in analysis of steroids in doping control.


PubMed | Institute of Doping Analysis and Sports Biochemistry
Type: Journal Article | Journal: Archives of toxicology | Year: 2012

Anabolic-androgenic steroids are frequently misused compounds in sports, and they belong to the controlled substances according to the requirements of the World Anti-Doping Agency. The classical techniques of steroid detection are mass spectrometry coupled to gas or liquid chromatography. Biological methods that base on the ability of substances to bind the steroid receptor are not applied in routine doping control procedures so far, but they appear to be useful for characterization of steroid androgenic potential. In this study we used the yeast androgen receptor reporter system (YAS), which in the past has already successfully been applied to both various androgenic substances and also urine samples. Giving attention to the androgenic potential of steroidal dietary supplements, we exemplified the analysis using both mass spectrometry techniques and the YAS-based assay on the product Syntrax Tetrabol which was a confiscated dietary supplement and marketed as a steroid precursor. Identification, structure and the kinetic behavior of its excreted metabolites were analyzed by NMR, GC-MS and LC-MS/MS. The androgenic potential of the parent compound as well as its metabolites in urine was evaluated with the help of the YAS. The application of urine samples with a previous deconjugation and the inclusion of urine density values were carried out and led to increased responses on the YAS. Further, the possibility of a complementary application of structure-based instrumental analysis and biological detection of androgenicity with the help of the YAS seems to be desirable and is discussed.

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