04. Anabolic Steroid Profiles

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04. Anabolic Steroid Profiles

 

CATEGORY: Anabolic Steroids 100 Courses

COURSE NUMBER: 04

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Syllabus

  • A Guide to Some Vitamins and Minerals & Supplements To Take
  • An Introduction Into Advanced Dieting
  • A complete guide to STEROID PROFILES
  • A Chart Comparing and Rating Different Steroids and Uses
  • How To Administer an Injection

What is Anabolic Steroid Profiles?

Steroid profiling in urine is a commonly employed method of clinical endocrinology, which is frequently used, for instance, for newborn screenings to detect enzyme deficiencies. The methodology was adapted and introduced in doping controls in 1983 by Donike et al. to allow for the determination of testosterone (T) misuse in sports, and later for the identification of the administration of related compounds. ‘The urinary steroid profile’ is composed of concentrations and ratios of various endogenously produced steroidal hormones, their precursors, and metabolites including T, epitestosterone (E), dihydrotestosterone (DHT), androsterone (And), etiocholanolone (Etio), dehydroepiandrosterone (DHEA), 5α‐androstane‐3α,17β‐diol (Adiol), and 5β‐androstane‐3α,17β‐diol (Bdiol). whereas the exact pathway leading to the formation of E has not yet been elucidated. The alteration of one or more of the concentrations of these parameters interferes with the naturally well‐balanced system and raises suspicion in routine doping controls either by increased or decreased concentrations and ratios. In order to improve the comprehensiveness and significance of this approach, an enclosure of additional parameters was recommended, which facilitates and supports the detection of surreptitious administrations of compounds such as T, E, or DHEA administration as well as bacterial activity. Therefore, androstenedione (precursor of T) and 6α‐OH‐androstenedione (metabolite of androstenedione), 5β‐androstane‐3α,17α‐diol (17‐epi‐Bdiol) and 5α‐androstane‐3α,17α‐diol (17‐epi‐Adiol), 3α,5‐cyclo‐5α‐androstan‐6ß‐ol‐17‐one (3α,5‐cyclo, metabolite of DHEA), or 5α‐androstanedione (Adion) and 5β‐androstanedione (Bdion) were utilized, respectively. Moreover, steroids with an origin independent of the androgen metabolism are monitored, serving as so‐called endogenous reference compounds (ERC) for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) in confirmatory analyses, which include, e.g. pregnanediol (PD), 11β‐hydroxy‐androsterone (11‐OH‐And), and 11β‐hydroxy‐etiocholanolone (11‐OH‐Etio). Additionally, the artificial formation of 19‐norsteroids and the endogenous production of boldenone (B) and B metabolites, which were observed in a few cases, are considered. A summary of steroids, their retention times, and characteristic fragment ions commonly monitored for steroid profiling are presented in Table using GC‐MS conditions as published elsewhere.

Steroid profiles, as determined routinely in doping control, provide essential information for several purposes as they are recorded, stored, and attributed to individuals incrementally with each collected sports drug testing sample. Hence, it is of utmost importance that laboratory standards are comparable within the group of World Anti‐Doping Agency (WADA)‐accredited drug testing sites to enable correlation of data obtained from one athlete on different occasions. This is guaranteed by comprehensive validations and proficiency tests, and the use of isotope‐dilution mass spectrometry (IDMS) employing certified reference material allows the precise and accurate evaluation of steroid profile data. Several studies demonstrated only small intraindividual variations of steroid profile parameters, especially within the ratios utilized for doping control purposes such as T/E, And/Etio, And/T, and Adiol/Bdiol. These ratios were not influenced by exercise or severe physical endurance performance, by menstrual cycle by circadian or annual rhythms. Hence, steroid profiles are valuable, for instance, for the management of elevated T/E ratios according to WADA guidelines. Longitudinal and retrospective evaluation of doping control samples in terms of steroid profiles enable the detection of abnormal alterations that trigger subsequent analyses such as confirmatory GC/C/IRMS measurements. Moreover, considerable long‐term stability of intraindividual steroid profiles support the detection of manipulation (e.g. urine substitution) in doping control urine sample collection and provide one of the most important parameters for specimen individualization in sports drug testing. A recent example showed that screening a database containing 14 224 urinary steroid profiles of athletes for specific values of four characteristic steroid ratios (T/E, And/Etio, And/T, and Adiol/Bdiol) yielded three samples that were all derived from one athlete. However, limitations of the prospects of steroid profiling for identification purposes were also reported recently. Robinson et al. described a case of seven doping control urine samples collected during a cycling stage race with moderately elevated T/E ratios. Different pattern classification tools were tested to categorize the most similar steroid profiles, but none of the models enabled a clear classification of the different urine samples. Finally, genetic profiling demonstrated that only three of seven samples originated from the same cyclist.

Nevertheless, the great importance of urinary steroid profiles and their utility in doping controls are manifold, and the information obtained is vital to the successful screening for steroidal anabolic agents. Thus, knowledge of factors that influence the steroid profile pattern is of central importance, in addition to parameters such as specific gravity, pH‐value, gender, sport discipline, and time of sampling; other factors such as pharmaceutical, technical, and biological issues need consideration when interpreting steroid profile patterns, which are outlined in the following text.

POPULATION BASED‐ VERSUS SUBJECT‐BASED REFERENCE RANGES FOR THE DETECTION OF TESTOSTERONE (T) MISUSE
In 1982, the test adopted by the International Olympic Committee (IOC) for detection of T administration was based on the T/E ratio. The T/E laboratory reporting threshold was derived empirically from an observed distribution of measurements in specimens collected from a large number of individuals and established at T/E = 6. With an adverse finding, it was mandatory to investigate the T/E results from previous and subsequent tests, i.e. assessing the T/E ratio intraindividually. The reason that elevated T/E ratios need a follow‐up before they are declared as adverse finding is the occurrence of naturally elevated T/E ratios. Since 2005, the WADA has changed the reporting threshold for T/E from 6 to 4 in order to improve the sensitivity for the detection of T misuse. The application of intraindividual T/E profiling was first discussed by Donike et al. in 1994. He demonstrated that subject‐based reference ranges react sensitively to variations and provide a better doping control approach than the ones utilizing population‐based reference ranges. Hence, subject‐based reference ranges of endogenous hormone concentrations or ratios such as T/E have been considered reliable tools to monitor various kinds of doping, employing endogenous steroids. Results of longitudinal urinary steroid profile studies outlined the low variability within the biosynthesis of endogenous steroids and that the metabolic pathway is in agreement with the stationary, homeostatic model for calculating subject‐based reference ranges.

In agreement with Donikes’ publications Sottas et al. improved this method recently by proposing the Bayesian screening test for the detection of abnormal values in longitudinal biomarkers. This test compares sequential measurements of a biomarker against previous readings performed on the same individual. The importance of such approaches was stressed by comprehensive studies concerning testosterone gel (T‐gel) administrations (vide infra). In a recent study 18 healthy male volunteers were treated for 6 weeks continuously and intermittently with T‐gel. The discriminating power of individual reference ranges was significantly superior to conventional population‐based reference ranges, especially for volunteers showing basal values for T/E less than 1. The mean of one participant’s pretest T/E ratio was 0.44. During the period of T‐gel application, all T/E values remained far below the cut‐off limit of 4, but all values were above the upper limit of the individual reference range.

Even now, in general, only atypical findings are followed up. The importance of having access to steroid profile data of all athletes’ samples in order to initiate further analytical tests (isotope ratio mass spectrometry = IRMS) or target doping controls may be a new possibility for steroid profiling in future doping control.

GENOTYPE‐DEPENDENT VARIATION OF T/E
T/E ratios vary among athletes of different ethnic origin. Typically, Asian people have lower urinary T/E values (<0.5) than Caucasians (approximately 1.0) and, thus, androgen doping exerts weaker effects on the T excretion in the Asian population, increasing the risk of false‐negative results. Recent studies described the lack of the UGT2B17 gene in some individuals. In 2006, a Swedish group investigated urine samples from 74 Korean and 122 Swedish men for T‐ and E‐glucuronides. The distribution of the natural logarithms of urinary T concentrations showed a distinct bimodal pattern in both groups, suggesting a monogenic inheritance. When the UGT2B17 genotypes were compared with urinary T levels, all the individuals homozygous for the UGT2B17 deletion genotype had no or negligible amounts of urinary T. This genotype was seven times more common in the Korean (66.7%) than in the Swedish population (9.3%). These data are in accordance with evaluations of reference ranges for Asian and Caucasian male and female athletes calculated from databases of the Asian Games 1994, the previous Asian Games 1990, and the routine doping control samples of Caucasian athletes measured in Cologne 1994 as well as with the results of the population‐based reference ranges of 5101 male and 1694 female athletes.

PREGNANCY
Pregnancy causes characteristic changes in the female steroid profile. Resulting from a missing feedback mechanism, the excretion of PD is significantly increased. The population‐based reference ranges of PD for females are between 80 and 3000 ng/ml. In the early stages of pregnancy between 5000 and 10 000 ng/ml of PD were detected, increasing to more than 20 000 ng/ml shortly before delivery. In addition, estrogenous substances are excreted in high amounts, with values increasing during the course of pregnancy and interfering with the internal standard used for the steroid profile evaluation. As a consequence, invalid results may be obtained.

19‐Norandrosterone is detected in the later course of pregnancy, probably formed as a side product during the conversion of steroids by aromatization. Urinary concentrations of 19‐norandrosterone during pregnancy were studied in five pregnant women, who collected morning urine samples once per week during the whole course of pregnancy. In addition, 50 spot urine specimens from different pregnant women were analyzed. The detection of 19‐norandrosterone was possible from the 14th week of pregnancy and the concentrations in healthy pregnant women showed an increase during the course of pregnancy with mostly less than 5 ng/ml. Only 7% of all samples showed higher concentrations with a maximum value of 16 ng/ml.

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