Biomedical subjects

D Thieme

Publications and source records attributed to D Thieme.

At least 19 recordsLinked to original sources

PubMed2005

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.

Administration, Cutaneous↗

PubMed2005

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.

Biotransformation↗

PubMed2004

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.

Administration, Oral↗

PubMed2003

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.

Adult↗

PubMed2001

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.

Atmospheric Pressure↗

PubMed2000

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.

Adrenergic beta-Antagonists↗

PubMed1999

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.

Adrenergic beta-Antagonists↗

PubMed1999

[Inhibition of 18F-FDG uptake in glioblastoma cells by FDG and 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.

Biological Transport↗

PubMed1995

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.

Anabolic Agents↗

PubMed1995

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.

Adrenergic beta-Agonists↗

PubMed1989

[Risk analysis and process control in food hygiene].

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.

Animals↗

PubMed1975