Sex differences in stress and immune responses during confinement in Antarctica.

BACKGROUND: Antarctica challenges human explorers by its extreme environment. The effects of these unique conditions on the human physiology need to be understood to best mitigate health problems in Antarctic expedition crews. Moreover, Antarctica is an adequate Earth-bound analogue for long-term space missions. To date, its effects on human physiology have been studied mainly in male cohorts though more female expeditioners and applicants in astronaut training programs are selected. Therefore, the identification of sex differences in stress and immune reactions are becoming an even more essential aim to provide a more individualized risk management. METHODS: Ten female and 16 male subjects participated in three 1-year expeditions to the German Antarctic Research Station Neumayer III. Blood, saliva, and urine samples were taken 1-2 months prior to departure, subsequently every month during their expedition, and 3-4 months after return from Antarctica. Analyses included cortisol, catecholamine and endocannabinoid measurements; psychological evaluation; differential blood count; and recall antigen- and mitogen-stimulated cytokine profiles. RESULTS: Cortisol showed significantly higher concentrations in females than males during winter whereas no enhanced psychological stress was detected in both sexes. Catecholamine excretion was higher in males than females but never showed significant increases compared to baseline. Endocannabinoids and N-acylethanolamides increased significantly in both sexes and stayed consistently elevated during the confinement. Cytokine profiles after in vitro stimulation revealed no sex differences but resulted in significant time-dependent changes. Hemoglobin and hematocrit were significantly higher in males than females, and hemoglobin increased significantly in both sexes compared to baseline. Platelet counts were significantly higher in females than males. Leukocytes and granulocyte concentrations increased during confinement with a dip for both sexes in winter whereas lymphocytes were significantly elevated in both sexes during the confinement. CONCLUSIONS: The extreme environment of Antarctica seems to trigger some distinct stress and immune responses but-with the exception of cortisol and blood cell counts-without any major relevant sex-specific differences. Stated sex differences were shown to be independent of enhanced psychological stress and seem to be related to the environmental conditions. However, sources and consequences of these sex differences have to be further elucidated.

Potential detection of low-dose transdermal testosterone administration in blood, urine, and saliva.

Administration of low amounts of endogenous hormones - so-called micro-dosages - are supposed to represent a major challenge in doping analysis. To model such a situation, we have studied transdermal administrations of 2.4 mg/24 h testosterone patches and examined various steroid concentrations in blood, urine, and saliva of 11 volunteers. Multiple samples were collected at t = 0, 3, 6, 9, 24, 48, and 72 h in four different phases, i.e., all combinations with/without physical exercise and with/without testosterone. Testosterone was analyzed by enzyme-linked-immuno-assay as well as by mass spectrometry and validated in an accredited anti-doping laboratory. Circadian controls with and without exercise did not provoke prominent alterations of whole, free, and salivary testosterone. Testosterone application for 24 h led to a significant (all p < 0.001) mean increase above controls: total testosterone (median: 5.2 vs. 8.0 ng/mL), free testosterone (median: 11.3 vs. 15.6 pg/mL), and salivary testosterone (median: 62.4 vs. 99.9 pg/mL). Additionally, all three testosterone measurements indicated significant correlations to each other (all r > 0.538, all p < .001). Circadian-matching showed peaking testosterone values after 6 h and 9 h, reaching highest augmentation up to 252.6 ± 123.5% in saliva after 9 h. After removal of the testosterone patch, all testosterone levels in blood, saliva, and urine returned to baseline within 24 h. Different techniques of hormone detection (enzyme-linked immunosorbent assay (ELISA), gas chromatography-tandem mass spectrometry (GC-MS/MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS)) indicated significant correlations. Results indicate that saliva, blood, and urine exhibit comparable hormone augmentation during micro-dose testosterone application, indicating a possible consideration in future doping analysis. The inter-individual variability was high in all biofluids, requiring the use of an individual biological passport rather than statistical values. Copyright © 2016 John Wiley & Sons, Ltd.

[Cardiovascular alterations associated with doping]. / Kardiovaskuläre Schäden durch Doping.

Doping -the abuse of anabolic-androgenic steroids in particular- is widespread in amateur and recreational sports and does not solely represent a problem of professional sports. Excessive overdose of anabolic steroids is well documented in bodybuilding or powerlifting leading to significant side effects. Cardiovascular damages are most relevant next to adverse endocrine effects.Clinical cases as well as forensic investigations of fatalities or steroid consumption in connection with trafficking of doping agents provide only anecdotal evidence of correlations between side effects and substance abuse. Analytical verification and self-declarations of steroid users have repeatedly confirmed the presumption of weekly dosages between 300 and 2000 mg, extra to the fact that co-administration of therapeutics to treat side-effects represent a routine procedure. Beside the most frequent use of medications used to treat erectile dysfunction or estrogenic side-effects, a substantial number of antihypertensive drugs of various classes, i.e. beta-blockers, diuretics, angiotensin II receptor antagonists, calcium channel blockers, as well as ACE inhibitors were recently confiscated in relevant doping cases. The presumptive correlation between misuse of anabolic steroids and self-treatment of cardiovascular side effects was explicitly confirmed by detailed user statements.Two representative fatalities of bodybuilders were introduced to outline characteristic, often lethal side effects of excessive steroid abuse. Moreover, illustrative autopsy findings of steroid acne, thrombotic occlusion of Ramus interventricularis anterior and signs of cardiac infarctions are presented.A potential steroid abuse should be carefully considered in cases of medical consultations of patients exhibiting apparent constitutional modifications and corresponding adverse effects. Moreover, common self-medications -as frequently applied by steroid consumers- should be taken into therapeutic considerations.

Urinary concentrations of ethyl glucuronide and ethyl sulfate as thresholds to determine potential ethanol-induced alteration of steroid profiles.

The suppression of steroid biotransformation resulting in a decrease of the major urinary metabolites--androsterone and etiocholanolone--and the elevation of testosterone/epitestosterone (T/E) ratios following ethanol administration is well described. At least the latter parameter T/E represents an important indicator for endogenous steroid abuse in doping control. The quantitative correlation between ethanol consumption markers and steroid profile alteration was evaluated, aiming to differentiate between permitted ethanol administration and potential steroid abuse. Steroid profiles, ethanol, ethyl glucuronide (EtG), and sulfate (EtS) were quantified after administration of ethanol (intended maximum ethanol concentration in blood was 1 mg/g) to 21 male and 15 female volunteers. EtG concentrations in urine (corrected by either specific gravity or creatinine concentration) were found to be most suitable for quantitative evaluations. Gender specific urinary EtG concentrations of 48 ug/ml (men) and 15.5 ug/ml (women) may be considered as useful thresholds for a potential ethanol-induced suppression of steroids biotransformation.

[Intraoperative consulting. An easy addition to the surgical safety checklist for perioperative quality assurance]. / Intraoperatives Controlling. Einfache Ergänzung der Checkliste zur perioperativen Qualitätssicherung.

BACKGROUND: It has been proven that pre-operative and postoperative surgical safety checklists designed to improve team communication and consistency of care are useful tools to improve surgical outcome. The aim of this study was to investigate the effectiveness of intra-operative consultation (IOC) with regard to its feasibility. IOC comprises an assessment of definite pathways in the operative procedure by another surgeon present in the operating theatre in a consultative capacity during specific steps of the operation. METHODS: Between January and December 2008 a total of 2,004 operations were evaluated according the IOC. Data on the frequency of the feasibility of IOC and on whether IOC led to decisions influencing the course of the procedure were analyzed. RESULTS: A total of 1,369 IOCs were carried out including regular IOC in 1,102 cases (80%) and tactical IOC in 267 cases (20%). In 90 cases (7%) consultation resulted in minor changes and in 100 cases (7%) major clinically relevant revision of the operative strategy was deemed necessary. CONCLUSION: It was found that IOC is feasible in the majority of operations. In the case of tactical IOC a large number of treatment-relevant decisions are taken. This means that in the area of operative disciplines IOC represents a potential preventive strategy within the framework of quality management and a useful addition to the WHO checklist to improve safety in surgery.

Application of discriminant analysis to differentiate between incorporation of cocaine and its congeners into hair and contamination.

The requirement to differentiate between incorporation and external contamination of drugs into hair is undisputed, in particular when dealing with compounds which are administered by sniffing or inhalation (e.g. cocaine). With the aim of making this discrimination, hair samples from cocaine (COC) users (group IN) and seized cocaine samples (group OUT) were compared regarding the parameters benzoylecgonine (BZE), ecgonine methyl ester (EME), ecgonine (ECG), anhydroecgonine methyl ester (AEME), cocaethylene (CE) and norcocaine (NCOC). Since most of these compounds may be minor by-products of COC or be formed by biotransformation or chemical degradation, the stability of each substance was carefully examined. COC was found to be converted into significant amounts of BZE, EME and ECG even under mild extraction conditions, while traces of NCOC proved to be a ubiquitous by-product of COC. Cocaine positive hairs and seized cocaine samples (diluted to relevant concentrations) were equally preprocessed and analyzed by LC-MS-MS. Out of the metabolites listed above, NCOC, CE and AEME (each normalised to COC) were significantly increased in the incorporation group (i.e. hair samples from cocaine users). Based on this approach, a statistical discriminant analysis enabled us to make a prediction (and estimation of uncertainty) for each cocaine positive hair sample as to its likelihood of belonging to the group of cocaine users or of being contaminated.

Detection of testosterone, nandrolone and precursors in horse hair.

Growing interest among several horse-breeder associations has initiated the development of a screening procedure to test for anabolic agents in hair, which has the advantage over blood and urine specimens of allowing long-term detection. An analytical method was established to monitor in tails or manes several anabolic substances available as veterinary medicines or as so-called nutritional supplements (clenbuterol, different esters or prohormones of nandrolone and testosterone). The analytical procedure to detect steroids in hair samples consists of the following steps: decontamination of the hair strand or segment with methanol/water (1:1), milling, extraction of the hair material in an ultrasonic bath using methanol, purification by liquid-liquid extraction (n-pentane/methanol, 25:1) and HPLC cleanup, derivatisation of the relevant LC fractions with MSTFA, and measurement using GC-MS/MS technique. The first objective of our study was the detection of exogenous nandrolone (nortestosterone, NT) in the horse hair; therefore nandrolone-associated compounds [nandrolone dodecanoate administered intramuscularly (i.m.) and a mixture of 4-estrenediol and 4-estrenedione, transdermal] were administered to four geldings. The highest concentrations of NT following i.m. treatment were measured after 10 days in a 2-cm hair segment (up to 18 pg/mg); NT was detectable for up to 120 days and in some cases up to 330 days in tail hair (limit of detection 0.3 pg/mg). Following transdermal application, nandrolone as well as the administered prohormones were identified in tail and mane until the latest sampling at 3 months. Furthermore, untreated stallions (128) were investigated to estimate the range of endogenous levels of NT and testosterone (T) in hair. Maximum values of 3 pg/mg (NT) and 1 pg/mg (T) were quantified originating from endogenous formation in the male horse. Additionally, a possible relationship between steroid concentrations in hair specimens and the age of stallions was appraised. NT and T were not detected in hair samples of control geldings. Following nandrolone treatment of geldings, highest values in hair exceeded the endogenous amount detected in untreated stallions. Therefore comparison of concentrations measured in control samples with the estimated endogenous levels could give a clue to exogenous application in cases of abnormally high amounts of NT or T. The possibility of the evaluation of threshold values is discussed as a means to verify an exogenous administration of NT and T in hair samples. Furthermore, the detection of a synthetic substance in hair, e. g. the parent steroid ester by itself, would be unequivocal proof of an exogenous origin of NT or T and the previous medication of the stallion.

Formation of 19-norsteroids by in situ demethylation of endogenous steroids in stored urine samples.

The formation of 19-norsteroids by demethylation of endogenous steroids in stored urine samples was observed. Suspicious urine samples (i.e. containing trace amounts of 19-norandrosterone and 19-noretiocholanolone) were selected and spiked with deuterated analogues of androsterone and etiocholanolone at concentrations corresponding to high endogenous levels (4 microg/mL). After incubation, respective 19-norsteroids (19-norandrosterone-d4 and 19-noretiocholanolone-d5) were identified in these samples by high-resolution mass spectrometry. The transformation of the 5 beta-isomer (etiocholanolone) yields about three-fold higher concentrations, compared to the 5 alpha-isomer. A significant temperature dependence was observed by comparison of reaction kinetics at room temperature (23+/-2 degrees C) and 37 degrees C. Concentrations of 19-norandrosterone-d4 and 19-noretiocholanolone-d5, respectively, were 2.7 and 3.6 times higher at elevated temperature. The conversion of androsterone-d4 to 19-norandrosterone-d4 did not exceed a relative amount of 0.1%. Incubation of the urine samples with androsterone-d4-glucuronide led to the production of 19-norandrosterone-d4-glucuronoide. A partial stabilization was observed after addition of metabolic inhibitors (e.g. EDTA). The application of the incubation experiments described may contribute to the clarification of adverse analytical findings regarding low levels of 19-norsteroid metabolites.

The beta-agonist clenbuterol in mane and tail hair of horses.

REASONS FOR PERFORMING STUDY: The beta2-agonist clenbuterol is commonly administered for therapeutic purposes in the horse, but its use an an anabolic agent is illegal. Clenbuterol can be detected in blood and urine for a relatively short period after administration and detection in hair could enhance the analytical range and be used to determine the history of clenbuterol application. HYPOTHESIS: That detection in mane or tail hair is possible over an extended period. METHODS: Four horses received 0.8 microg clenbuterol hydrochloride/kg bwt b.i.d. for 10 days. Four other horses were used as untreated controls. Blood, urine, mane and tail hair samples were taken on Day 0 (before) and 5, 10, 30, 35, 40, 60, 90, 120, 150 and 360 days after start of treatment. Gas chromotography/high resolution mass spectrometry (GC/HRMS) was developed for clenbuterol analysis: limit of detection was 0.2 pg/mg; intra-assay repeatability limit r = 0.06 (confidence level 95%); interassay repeatability limit r = 0.03 (confidence level 95%). Prior to treatment, clenbuterol was absent from all samples analysed. RESULTS: Clenbuterol was detectable as early as Day 5 in tail and mane hair of Segment 1 (0-20 mm from the roots) and was maximal on Day 90. However, as time progressed, shift into lower 20 mm segments was observed. On Day 360, the maximum concentration (up to 21 pg/mg) was located in Segment 13, i.e. 26-28 cm from roots of hair. Clenbuterol was not detectable in blood or urine after Day 30. Mane and tail hair results were very similar. CONCLUSIONS: The study showed that the beta-agonist clenbuterol can be found in mane and tail hair of horses after extended periods. POTENTIAL RELEVANCE: It will be possible to detect clenbuterol in breeding and show horses where anabolic drugs have been used illegally to improve conformation. This method may also be helpful to monitor therapeutic clenbuterol treatment.

Potential parameters for the detection of hGH doping.

The aim of our hGH application study with non-competitive athletes was the investigation of selected serum parameters from different processes affected by hGH. Fifteen athletes (age 21-33, mean 24) were treated with 0.06 IU hGH/kg BW per day or placebo (10 hGH, 5 placebo) respectively for 14 days. Blood samples were taken prior to, during and until 10 weeks after treatment. The concentrations of the following markers were determined in relevant serum samples: IGF-I, IGFBP-3, ALS, PIIINP, PINP, osteocalcin, and leptin. The IGF-I concentration increased rapidly within the hGH treatment group and showed significantly higher levels compared to baseline even 3 days after application. The response of the IGFBP-3 to the hGH applications was lower in comparison to IGF-I. The hGH group showed an increasing IGFBP-3 compared to baseline from day 4 till day 15. The response of PIIINP to hGH is clearly delayed compared to the IGF-I axis, but the PIIINP concentration remains on an increased level for a longer period (from day 4 until day 21). The time course and the extent of response varied strongly interindividually. PINP and osteocalcin showed only a small response to hGH applications. These parameters are characterised by a strong scattering of base values compared with the small response. In the hGH treatment group very different leptin concentrations were found at the beginning of the study, but after treatment decreasing leptin levels were observed in all cases. The determination of only one parameter will not be sufficient for detection of hGH abuse. A combination of markers by mathematical methods can be helpful to distinguish between placebo and hGH-treated athletes. By using the suggested discriminant function the data sets of hGH and placebo-treated athletes could be separated without false positive results.

Screening, confirmation and quantification of diuretics in urine for doping control analysis by high-performance liquid chromatography-atmospheric pressure ionisation tandem mass spectrometry.

A sensitive, selective, robust and fast method to identify 32 diuretics and masking agents in urine is described. The analytical procedure is reduced to a single XAD extraction step for sample preparation, followed by reversed-phase liquid chromatography in combination with atmospheric pressure ionisation/tandem mass spectrometry. This technique is, after minor modifications, suitable for screening analyses and confirmation of identity as well as quantitation of diuretics. Considerations relating to the stability and metabolism of the compounds are given if relevant for routine screening analyses.

Analytical strategy for detecting doping agents in hair.

Lists of banned classes of doping agents are released by the International Olympic Committee, adopted by other sports authorities and updated regularly, including the substance classes stimulants, narcotics, diuretics, anabolic agents, peptide hormones, beta-blockers etc. There are different classes of restriction: anabolic and masking agents (anabolic steroids, diuretics etc.) are always banned for athletes regardless of their topical activity (training or competition) several substances are permitted with certain restrictions (caffeine below a cut-off value, or inhalation of some beta 2 agonists) beta-blockers are prohibited in competitions of certain sports disciplines the majority of the substances (stimulants, narcotics etc.) is prohibited during competitions, so that they do not have to be analysed in out-of-competition samples. A differentiation between training and competition period is impossible by means of hair analysis due to the uncertainty of (especially short-term) kinetic considerations related to hair growth. Therefore, the analytical identification of doping relevant substances in hair is not always a sufficient criterion for a doping offence and the identification of stimulants, beta-blockers etc. in hair would be entirely irrelevant. The most interesting target substances are certainly the anabolic agents, because their desired action (enhanced muscle strength) lasts longer than the excretion, leading to sophisticated procedures to circumvent positive analytical results in competition control. Besides the analysis of out-of-competition control samples, the long term detection of steroids in hair could provide complementary information. An analytical approach to the identification of exogenous steroids in hair requires consideration of the presence of many other steroids in the hair matrix interfering the analysis at trace levels, and of a limited chemical stability. The analysis of endogenous steroids in hair appears to be even more complicated, because the possibility of many biotransformation reactions from (into) other precursors (metabolites) has to be taken into account. Precursor substances of anabolic steroids (especially esters as application forms) are very promising analytical targets of hair analysis, because they can only be detected after an exogenous intake. The quantitative evaluation of active parent compounds like testosterone (which is actively involved in physiological processes of hair growth) in hair is still controversial. Clinical applications under reproducible conditions can be useful, but the biovariability of these parameters will probably prevent the definition of acceptable cut-off levels as a criterion of abuse.

[Inhibition of 18F-FDG uptake in glioblastoma cells by FDG and glucose]. / Hemmung der 18F-FDG-Aufnahme in Glioblastoma-Zellen durch FDG und Glucose.

It was the aim of the study to compare the inhibition of 18F-2-Fluor-D-deoxy-glucose uptake (18F-FDG) in tumor cells by various concentrations of FDG carrier or D-glucose in an experimental model using tissue culture and positron emission tomography (PET). Glioblastoma cells in culture were incubated with 18F-FDG with and without added carrier or in presence of glucose concentrations in the range from 0-5 mmol/L. Cellular uptake of 18F-FDG was measured after 20 min. of incubation in PBS-buffer containing different sugar concentrations. The uptake was determined with a PET camera. The similarity of the kinetics of the FDG and glucose uptake are backing the hypothesis that both substrates use the same carrier system. The more intense inhibition of the 18F-uptake by FDG can be explained by the different intracellular metabolism of both substrates. The results explain the clinical experience that there is an optimal 18F-FDG uptake in the patient's tumor when the blood glucose level is as low as possible and the specific activity of 18F-FDG is very high.

Introduction to the application of capillary gas chromatography of performance-enhancing drugs in doping control.

Performance-enhancing drugs banned by antidoping rules are detected in doping control preferably by hyphenated chromatographic techniques, capillary gas chromatography in particular. Based on the prohibited classes of substances and on the general aspects of sample collection and preparation, a survey is given about the usual procedures of screening, identification and confirmation of the most important doping agents: stimulants, narcotics, anabolics, diuretics, beta-blockers. In addition to gas chromatography itself, the application of various MS techniques doping is outlined.

Chromatographic techniques--the basis of doping control.

The principal definition of doping, the groups of banned compounds and the basic analytical problems and strategy of doping analysis are outlined, and the position of chromatography in doping analysis is explained. Examples of the application of GC-MS, especially high-resolution MS. and of LC-thermospray MS to doping problems are given. A practical case is presented briefly, showing the post-analytical problem of evaluating even unequivocal results.

An improved method for clenbuterol screening using high resolution selected ion recording.

A method has been developed using reliable GC derivatization techniques interfaced with a high resolution mass spectrometer. The method has proved successful for the detection of low levels (less than 1 ppm) of clenbuterol in complex biological matrices. Selected ion recording of two characteristic isotopic fragment ions provides a specific mode of detection by verifying the GC retention time of these ions and also by comparing their relative abundance. Analysis of urine samples demands higher mass spectrometric resolution, and 40 000 (10% valley) was found to be a prerequisite for accurate integration of the drug-related chromatographic peaks. The method developed is suitable for adaptation to a completely unattended automated routine incorporating sample injection, storage and retrieval of source tuning parameters, and data processing.

[Risk analysis and process control in food hygiene]. / Lebensmittelhygienische Risikoanalyse und Prozesskontrolle.

It is introducing and recommending the systematic utilization of complexes of measures for food hygienic process control (good manufacturing practice--hazard analysis--critical control points), which are in international discussion.