Testosterone Concentrations In Samples Research Articles

Testosterone Doping: Dealing with Genetic Differences in Metabolism and Excretion

The first documented use of testosterone as a performanceenhancing substance in sport began in the 1950s. The success of Russian weightlifters during this era led Dr. John B. Zeigler to surreptitiously obtain information from their trainers that they were using testosterone. Later, Zeigler would use Dianabol to enhance the performance of weightlifters at the York Barbell Club beginning in 1958. Once Pandora’s Box was open, it was impossible to close. The growth and widespread use of anabolic steroids in many competitive sports eventually led the U.S. Congress to schedule testosterone and other anabolic agents under the Anabolic Steroids Control Act in 1990 (Public Law 101647). For many years, the medical community argued that there was no proof that testosterone enhanced athletic performance. Finally, in 1996, Bhasin and co-workers showed that supraphysiological doses of testosterone indeed produced performance benefits (1), something the athletes had known for four decades. The urinary testosterone to epitestosterone (T/E) ratio was the first test to be used for evaluation of abuse of a natural (or endogenous) substance such as testosterone to enhance performance in sport. The production of reliable quadrupole gas chromatograph/mass spectrometers in the mid-1970s allowed more widespread study of urinary steroid profiles. Donike and coworkers (2) observed an unusual distribution of testosterone concentrations in samples collected at the Moscow Olympics in 1980. In 1982, Donike proposed the use of the urinary T/E ratio based on the observation that epitestosterone was excreted in the urine in a relatively constant amount and as such constituted a way to compensate for the variability in testosterone concentration that occurred as a function of the diluteness of the urine (2). Because the primary metabolite of testosterone is testosterone glucuronide, the procedure developed by Donike used -glucuronidase in the preparation of the samples for analysis. In 1983, the International Olympic Committee accepted a T/E ratio of greater than 6:1 as evidence of testosterone doping, based on the 99th percentile of the log normal distribution of ratio values established by Donike and his co-workers. It was soon clear that the T/E ratio based on population reference ranges had limitations. For example, it had been noted in 1970 that genetic make-up could have a significant effect on urinary testosterone excretion, whereas epitestosterone excretion was unaffected (3). As population T/E ratio data accumulated in the anti-doping laboratories, it was clear that there were two distributions of urinary T/E ratios in both men and women, one with a maximum frequency at a T/E ratio of about 0.1 and a much larger population with a maximum frequency at a T/E ratio of about 1. Within the anti-doping community, the smaller population was referred to as low mode, and it was known that administration of testosterone to low-mode individuals did not result in a T/E ratio above the population threshold (4). Jakobsson and her colleagues (5) provided the mechanism for lowmode excretion in 2006 when they demonstrated that the UGT2B17 gene deletion was the cause of significant differences in testosterone glucuronide excretion between Korean and Swedish men. In their current paper in this issue, JakobssonSchulze et al. (6) report that when subjects deficient in the UGT2B17 gene (del/del) receive exogenous testosterone, their T/E ratio does not rise above the current population-based threshold of 4:1. One potential route of excretion of testosterone in UGT2B17 del/del genotypes is through other phase II metabolites such as testosterone sulfate. Borts and Bowers (7) directly measured the concentrations of testosterone and epitestosterone sulfate and glucuronide using HPLC/tandem mass spectrometry. Low-mode excretors did not produce larger than normal amounts of testosterone or epitestosterone sulfate. There was also no change in the relative production of sulfate conjugates. Because phase II metabolism does not explain the difference in testosterone handling in these individuals, it would be interesting to know what phase I metabolism products are increased in individuals with the UGT2B17 del/del polymorphism. This information may help develop a more sensitive test for testosterone

Application of enzyme-linked immunosorbent assay (ELISA) for determining testosterone levels in the follicular fluid of prepubertal and pubertal heifers

An enzyme-linked immunosorbent assay (ELISA) was used to measure follicular fluid testosterone concentrations in small size antral follicles (less than 5 mm in diameter) obtained from the ovaries of prepubertal (4 months old) and pubertal (1–2 years old) heifers. The homologous ELISA was developed by raising specific antibodies against testosterone in New Zealand White rabbits, using testosterone-3 carboxy methyl oxime (CMO): bovine serum albumin (BSA) as immunogen, and by labelling testosterone-3 CMO with horseradish peroxidase. The standard dose-response curve covered the range between 0 and 1 ng per well with follicular fluid analysed without previous extraction. Sample interferences were minimised by dilution. The intra- and inter-assay coefficients of variation were less than 7%. Recovery of added testosterone averaged 96.9 ± 1.2% and the minimum limit was 0.4 pg per well with sensitivity of 50% binding at 81.7 pg per well. A comparison with radioimmunoassay had a correlation of 0.98. Mean ± SE follicular fluid testosterone concentrations in samples from prepubertal heifers were 54.81 ± 5.85 ng ml −1 and in pubertal heifers 56.94 ± 5.49 ng ml −1. These concentrations are typical for testosterone-rich follicles. This ELISA is simple and direct and can be used for determining follicular fluid testosterone concentrations in cattle.

Sex hormone concentrations in blood serum from the North Atlantic fin whale (Balaenoptera physalus)

Blood serum concentrations of testosterone and progesterone were measured in postmortem samples taken at sea from 814 fin whales (Balaenoptera physalus) caught during the summers (June-September) of 1981-1989. The ages of 781 of these animals were also assessed. The testosterone concentrations in samples from 352 males averaged 2 nmol/l; 41 samples had concentrations of 0.1 nmol/l or lower and 34 of these came from whales aged between 2 and 14 years and showed a Gaussian type of age distribution with a peak number at 7 to 8 years. The mean testosterone concentrations in the males increased by more than fourfold between June and August. Serum progesterone concentrations of the 462 females fell into three separate groups: (1) group I with values < or = 0.1 nmol/l; (2) group II with intermediate values of > 0.1 nmol/l but < 10 nmol/l; (3) group III with values of > or = 10 nmol/l. These three groups of females seemed to consist respectively of young sexually immature females, mature non-pregnant females and pregnant females. The age distribution in the groups indicated that puberty in females is attained chiefly between the ages of 7 and 10. The yearly pregnancy rate (that percentage of all females caught and studied in a year which had progesterone values > or = 10 nmol/l) was between 35% and 55%, except in 1987 when it was 67%. The yearly pregnancy rate would range from 56% to 93% if only mature females (i.e. those with serum progesterone > 0.1 nmol/l) were considered.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that the seasonal antler cycle of adult Reeves' muntjac (Muntiacus reevesi) is not associated with reproductive quiescence

Data were obtained from post-mortem investigations of 190 culled and road-killed muntjac bucks between 1967 and 1989. Although adult bucks (i.e. those having undergone at least one antler cycle) have a synchronous annual antler cycle, unlike temperate-zone cervids there was little seasonal variation in testis size or activity, or in the size and activity of the epididymidis or accessory reproductive glands. Spermatogenesis was not abated when the antlers were in velvet and year-round fertility was achieved without additional sperm storage. There was little seasonal change in plasma testosterone concentrations in samples obtained from captive and free-living bucks although castration caused antler casting and prevented mineralization. Hence the data are equivocal as to the role of steroids in driving the antler cycle; experimental work on this species would be valuable in examining the mechanisms which regulate the antler cycle.

Canine testosterone concentrations in samples stored at room temperature.

Paired samples of plasma and whole blood from 10 healthy male dogs were left on the laboratory bench for periods of time up to one week before being stored at −20 °C. Testosterone concentrations in these samples were measured at a convenient time a few weeks later. The effect of storage at room temperature was to increase the concentrations of testosterone in both plasma and whole blood. However, the changes in plasma concentrations were insignificant up to 144 h whereas the increases in whole blood were significant after 48 h.

A sensitive enzymeimmunoassay with a fluorimetric end-point for the determination of testosterone in female plasma and saliva

A fluorimetric enzymeimmunoassay has been developed having the sensitivity (50O fg/assay tube) required for determining testosterone concentrations in female plasma and saliva samples. The assay featured a solid-phase antiserum raised against an llα-hydroxytestosterone-11-hemisuccinate bovine serum albumin conjugate, an llα-hydroxytestosterone-11-hemisuccinate horseradish peroxidase conjugate as the “enzyme label” and p-hydroxyphenylacetic acid as the substrate for the development of fluorescence. Specificity was ensured by “extracting” testosterone from samples with a solid-phase anti testosterone-3-¦O-carboxymethyl¦-oxime serum. The assay was shown to satisfy accepted validation criteria providing results in good agreement with routine radioimmunoassay procedures in both plasma (r > 0.98, n = 28) and saliva (r > 0.99, n = 28). In saliva samples collected at 2 hourly intervals by normal healthy women (n = 5) testosterone concentrations showed a well defined circadian rhythm: the mean testosterone concentration in early morning samples (174 pmol/litre) fell by 83% in late evening collections. In healthy female volunteers (n = 7), mean salivary testosterone concentrations in samples collected daily throughout one complete cycle ranged from 5O to 218 pmol/litre. Following dexamethasone administration testosterone concentrations in plasma fell by approximately 50% and salivary concentrations were undetectable after one hour. This enzymeimmunoassay may be useful in studies of female infertility.

Amniotic fluid testosterone: Implications for the prenatal diagnosis of congenital adrenal hyperplasia

The concentration of testosterone in amniotic fluid was measured in ten samples obtained between 16 and 36 weeks' gestation from a pregnancy in which the fetus was subsequently found to have congenital adrenal hyperplasia due to 21-hydroxylase deficiency and in 48 control samples collected between 12 and 39 weeks' gestation. The mean testosterone concentration in control samples was 21.2±14.6 (S.D.) ng. per 100 ml. (range 3.2 to 67.0 ng.per 100 ml). There was no sex difference or significant change in amniotic fluid testosterone concentration with advancing gestation. The testosterone concentrations in samples from the pregnancy in which the fetus was affected were within the range found in samples from control pregnancies. The concentration of testosterone in amniotic fluid was measured in ten samples obtained between 16 and 36 weeks' gestation from a pregnancy in which the fetus was subsequently found to have congenital adrenal hyperplasia due to 21-hydroxylase deficiency and in 48 control samples collected between 12 and 39 weeks' gestation. The mean testosterone concentration in control samples was 21.2±14.6 (S.D.) ng. per 100 ml. (range 3.2 to 67.0 ng.per 100 ml). There was no sex difference or significant change in amniotic fluid testosterone concentration with advancing gestation. The testosterone concentrations in samples from the pregnancy in which the fetus was affected were within the range found in samples from control pregnancies.