Urinary Testosterone Research Articles

Hyperandrogenism and Hyperinsulinism in Children of Women With Polycystic Ovary Syndrome: A Controlled Study

Both hyperandrogenism and insulin resistance are heritable family traits that may cluster in children whose mothers have polycystic ovary syndrome (PCOS). This case control study compared reproductive and metabolic abnormalities in 32 healthy children, 17 girls and 15 boys, and 38 children whose mothers did not have PCOS. The children ranged in age from 4 to 14 years. There were no differences between PCOS and control children in the length of gestation or birth weight. Urinary levels of luteinizing hormone were significantly lower in Tanner IV-V PCOS girls than in control subjects. Urinary testosterone levels were significantly greater in Tanner II-III PCOS boys than in controls. No significant differences in levels of dehydroepiandrosterone (DHEA) or DHEA sulfate were observed. Fasting insulin levels did not differ significantly between PCOS and control children of either gender, whether saliva or blood samples were analyzed. Nevertheless, mean area-under-the-curve salivary insulin levels were significantly higher in Tanner IV-V PCOS girls in the later stages of puberty than in control children. Five of 15 PCOS children met the de Ferranti criteria for metabolic syndrome, compared to only one of 12 control children. No children in either group met criteria for abnormal fasting blood glucose. Although ovaries tended to be larger in PCOS girls, none of the children examined by transabdominal ultrasonography had evidence of polycystic ovaries based on morphological criteria. This study suggests that hyperinsulinism may be a familial feature of PCOS children - at least girls - but it does not emerge until the later stages of puberty. It is possible that reproductive abnormalities characteristic of PCOS will develop later in life.

Enzyme Immunoassay of Testosterone, 17β-Estradiol, and Progesterone in Perspiration and Urine of Preadolescents and Young Adults: Exceptional Levels in Men's Axillary Perspiration

Enzyme immunoassays for testosterone, 17beta-estradiol, and progesterone were validated for human facial and axillary perspiration and compared to levels in urine. In study 1, these assays were applied to samples from preadolescent girls and boys and young women and men. Men's axillary perspiration contained substantially higher levels of steroids than seen in other substrates from men or in any sample from women, boys, and girls. Male axillary steroid levels were very variable across individuals, and on average they exceeded levels in facial perspiration by 90-fold for testosterone and 45-fold for estradiol. Men's urinary testosterone also exceeded urinary levels of the other subjects. In study 2, axillary perspiration, urine, and saliva were collected from young men. Substantial axillary levels of testosterone and estradiol were again observed. Correlations of the same hormone among the different substrates were generally very low, except for a small correlation between estradiol levels measured in axillary perspiration and urine in study 2. High unconjugated steroid content in men's axillary excretions could, if absorbed by women during intimacy, be implicated in pheromonal activity.

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

Quantitative confirmation of testosterone and epitestosterone in human urine by LC/Q‐ToF mass spectrometry for doping control

Testosterone (T) is the primary male sex hormone. In addition to the development of secondary sex characteristics, testosterone has anabolic effects including increases in muscle size and strength and increases in lean body mass, making it an attractive candidate to enhance athletic performance. In the case of exogenous administration of testosterone, the ratio of testosterone to its isomer, epitestosterone (E), is elevated. WADA has set a standard for T/E ratios of 4.0 as indicative of possible exogenous testosterone administration. Typically, a sample that screens for a T/E ratio above that threshold is then subjected to quantitative confirmation by GC/MS. This methodology, however, can limited due to sensitivity issues as well as a limited number of qualifying ions that can be used for unambiguous identification. We have developed a confirmation method which uses liquid/liquid extraction, followed by room temperature Girard P derivatization, and analysis using LC/MS-ToF. We observe a number of advantages over conventional GC/MS analysis. Analysis time is decreased. Sensitivity is increased, resulting in limits of detection of 2 and 0.5 ng/ml for testosterone and epitestosterone, respectively. The number of diagnostic qualifier ions is also increased allowing more confident identification of the analytes. Finally, while this method has been developed on a QToF instrument, it should be easily transferable to any tandem LC/MS/MS system.

Measuring urinary testosterone levels of the great apes—Problems with enzymatic hydrolysis using Helix pomatia juice

Helix pomatia ( Hp) juice is a common enzymatic preparation for deconjugation of urinary steroids. It has been used in many published studies on urinary testosterone (T) in chimpanzees and bonobos, although the ability of Hp juice to convert other urinary steroids into T has been reported for human urine. We developed a protocol for determination of reliable T levels in primate urine using liquid chromatography–mass spectrometry. T levels were determined in a set of human, bonobo and chimpanzee urine samples (A) by measurement of intact testosterone glucuronide (TG) and testosterone sulfate (TS), (B) after hydrolysis/solvolysis with β-glucuronidase from Hp and (C) from Escherichia coli. When samples were hydrolyzed with Hp juice, results were not correlated with the direct assay of TG and TS, and determined T concentrations were considerably higher. By contrast, hydrolysis with E. coli β-glucuronidase yielded a good agreement of T concentrations. We demonstrated the ability of Hp juice to convert androst-5-ene-3β, 17β-diol (A 5diol) into T using commercial standards and within the urine of all three species. As A 5diol usually is present at higher levels in urine than T, this artifact leads to erroneous results for T concentrations in primate urine. The proportion of T excreted as sulfate (TS) is often neglected as TS can only be cleaved by additional solvolysis. In all three species, we found substantial amounts of TS in the urine of some subjects and a high variance of TS proportion between and within subjects. Therefore the inclusion of solvolysis into the sample preparation seems necessary.

Isolation and quantification by high-performance liquid chromatography–ion-trap mass spectrometry of androgen sulfoconjugates in human urine

Together with steroid glucuronides, sulfoconjugates may be used as markers of steroid administration as well as endogenous steroid production. A fast and sensitive analytical procedure has been developed for the simultaneous separation, determination and quantification of sulfate and glucuronide derivatives of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio) and dehydroepiandrosterone (DHEA) in human urine. First, a weak anion-exchange solid-phase extraction support (SPE Oasis WAX) was used for complete and rapid separation of sulfates and glucuronides in two extracts after loading of urine sample (2 mL). Then sulfates were analyzed directly by high-performance liquid chromatography–ion-trap mass spectrometry (LC–MS/MS) with electrospray ionization in negative mode. Chromatographic separation of the targeted sulfoconjugates was achieved using a Waters XBridge C 18 column (150 mm × 4.6 mm I.D., 5 μm) with gradient elution. Assay validation demonstrated good performance for instance for T sulfate (TS) and E sulfate (ES) in terms of trueness (89–107%), repeatability (3.4–22%) and intermediate precision (5.8–22%) over the range of 2–200 ng/mL (corresponding to 1.5–147 ng/mL as free steroids). Results obtained on biological samples demonstrated the suitability of this analytical strategy for direct measurement of androgen sulfoconjugates and glucuroconjugates in human urine.

Evaluation Of High Level Tennis Players In A Competition By Measuring The Androgens/Corticosteroids Urinary Ratio

Tennis is an intermittent intensity sport with an average intensity during matches of 55 - 65 % VO2 max and with a duration of 1 - 5 hours in high level tennis players. Physical activity generates stress situations which can be appreciated as hormonal changes depending on effort intensity and duration. Urinary hormones and their metabolites have been used in order to evaluate physical effort, but more knowledge is needed regarding this topic. PURPOSE: The aim of this study is to evaluate physical effort generated during an official match in high level tennis players by using different urinary anabolic / catabolic hormone ratios and to determine which of them could be the best indicator of fatigue analysis. METHODS: Eight professional tennis players, ranked in the top 100 ATP, performed this study (Age: 24,88 ±2,59; Height: 1,80 ±0,05; Weight: 75,63 ±5,21). Urine samples were collected before (60 min before competition) and after (60 min after competition) a tournament match (quarterfinals corresponding to the Spanish Tennis Master). The matches started at 11 a.m and they had an average duration of 90 ± 18 min. Urinary testosterone (T), DHEA (D), cortisol (C), T/C ratio and DHEA/C steroid ratio were determined by using chromatographic techniques for their analysis: GC/MS and GC/MS/MS. The density of urinary samples was in the range of 1,005 - 1,025 g/mL, and the pH was in the normal range of 4,7 - 7,8. The creatinine levels of all the samples were determined in order to report the steroid concentrations related to this urine parameter. The creatinine concentration was determined by spectrophotometry using Sigma Kit "Creatinine 555 - A&". The Wilcoxon test was performed for statistical analysis. Ap value of <0.05 was used to determine statistical significance. RESULTS: The DHEA/C ratio decreased (2,97 ± 0,46 to 0,77 ± 0,26, p<0,05), T/C ratio decreased (1,04 ± 0,35 to 0,53 ± 0,11) although statistical significance was not reached. These ratio changes were due to a decrease in urinary androgens excretion as well as an increase in urinary corticosteroids excretion. A bigger decrease in suprarenal androgens may indicate a greater implication of this glandule in the early phases of recovery. CONCLUSION: The DHEA/C urinary ratio could be a useful parameter in order to gain information on physical effort and fatigue in high level tennis players.

Hyperandrogenism and Hyperinsulinism in Children of Women with Polycystic Ovary Syndrome: A Controlled Study

Hyperandrogenia and insulin resistance are heritable family traits, likely to cluster in children of polycystic ovary syndrome (PCOS) mothers. We performed a case control study of PCOS children (n = 32) compared with children from control women (n = 38) for reproductive and metabolic abnormalities, stratifying results by three Tanner stage groupings. The children underwent history and physical examinations, a 3-h timed urine collection, a 2-h oral glucose tolerance test, and abdominal ultrasound examination (females only). Serum was obtained in older children (age > 8 yr) who consented. Urine LH levels were significantly lower in the Tanner IV-V PCOS girls compared with controls (P = 0.04). Urine testosterone levels were significantly elevated in Tanner II-III PCOS boys compared with controls (P = 0.007). There were no significant differences in dehydroepiandrosterone levels. We validated the correlation between salivary and serum levels of insulin (insulin areas under the curve) in an adult population [n =30, Pearson correlation coefficient (r) = 0.67; P < 0.0001], which also replicated in the children (2-h insulin r = 0.57; P = 0.0004). Mean area under the curve salivary insulin levels were significantly higher in the Tanner IV-V PCOS girls in the later stages of puberty when compared with controls (3625 +/- 1372 vs. 1766 +/- 621 min x muU/ml, 95% confidence interval 475-3242; P < 0.02). Hyperinsulinism may be a familial characteristic of PCOS children (or at least girls) but does not appear until the later stages of puberty. Other reproductive abnormalities that characterize PCOS may develop later.

Assessment of urinary total testosterone production by a highly sensitive time‐resolved fluorescence immunoassay

Radioimmunoassay (RIA) that involves purification of the analyte by organic solvent extraction is widely used. Although the extraction RIA is reliable when properly validated, it is time consuming and radioactive, we measure urinary total testosterone with a highly sensitive rapid and accurate time-resolved fluorescence immunoassay (TRFIA) method. High affinity antitestosterone antibody and Eu(3+) labeled Donkey antisheep IgG as tracers were used. The assay was evaluated for specificity, sensitivity, analytical recovery, precision and dilution linearity by the TRFIA method on urine samples. A satisfactory standard curve for testosterone TRFIA has been developed with good sensitivity (5.1 pmol/L). The validity of the assay for urinarytotal testosterone was confirmed by the good correlation between the results obtained by TRFIA (X) and those RIA (Y) (Y=0.075+0.971X, R=0.992). Specificity, analytical recovery, precision and dilution linearity studies were determined and all found to be satisfactory. Male urinary total testosterone excretion ranged from 64.00 to 374.11 nmol/24 hr, which was about four times more than the range for women urinary testosterone excretion (14.16-100.65 nmol/24 hr), which suggests that a direct, reliable, easy to automate, highly sensitive and specific TRFIA type assay for the measurement of total testosterone in urine samples has been developed.

Seasonal Changes in Urinary Prostate‐Specific Antigenic Activity in Male Japanese Macaques (Macaca fuscaa fuscata)

Prostate-specific antigen (PSA) is usually detected in male adult urine and semen according to the Tanner stage development of males from birth to adolescence. To further study the pituitary-testicular axis in males, we determined urinary PSA levels in primates. Urinary PSA was detected with the use of anti-human PSA monoclonal antibody in male adult Japanese macaques (Macaca fuscaa fuscata) of seasonal breeding status. PSA activity in aseasonal animals (crab-eating macaques, Macaca fascisularis) did not change throughout the year; however, alterations in PSA activity were observed in Japanese macaques during breeding season, with the highest levels observed between October and January, the lowest levels between January and June, and a gradual increase in PSA activity observed from August until October. Although primate urinary PSA produces 2 polypeptide bands of approximately 55 and 33 kd, in addition to a band corresponding to human urinary PSA, the 33-kd polypeptide band was less pronounced during nonbreeding season in Japanese macaques. Urinary testosterone (T) levels in seasonally breeding animals (Japanese macaques) changed in parallel with urinary PSA levels. When urinary PSA and T levels were compared among animals during the breeding season (from October to February) and the nonbreeding season (from March to September), significantly increased PSA and T levels were observed during the breeding season. Furthermore, PSA and T levels in a monkey housed in a cage placed between 2 female cages were elevated compared with other monkeys. Increased PSA activity was observed concurrent with menstrual blood loss in females. These results suggest a link between PSA activity and testosterone levels, which could be influenced by changes in the female menstrual cycle.

Microscale immunoaffinity SPE and MEKC in fast determination of testosterone in male urine

Conventional methods for the determination of testosterone in body fluids typically suffer from poor recovery, lack of specificity, complex sample pretreatment, or the need for derivatization. Here, a simple, specific, and fast analysis method for testosterone was developed, with a methodology based on testosterone-specific immunoaffinity SPE (IA-SPE) and subsequent analysis by partial filling MEKC (PF-MEKC). An immunosorbent consisting of a recombinant antitestosterone Fab fragment covalently attached to activated Sepharose was prepared. IA-SPE and PF-MEKC were set up in hyphenated and off-line constructions, and the applicability of the two constructions in analysis of testosterone in male urine was investigated. The results obtained with the hyphenated construction proved to be only indicative of the presence of testosterone. The off-line IA-SPE and PF-MEKC construction, however, was successfully used in the determination of free testosterone in male urine samples after enzymatic hydrolysis of the glucuronide conjugates. Except for the hydrolysis reaction, no sample pretreatment was required. After hydrolysis, the overall analysis time per sample was only 14 min. The off-line IA-SPE and PF-MEKC method proved to be robust, sensitive (LOQ 35 mug/L), and specific, enabling separation of testosterone from four related steroids. Thus, it provides attractive features when compared to traditional methods for determination of testosterone in male urine.

Deletion polymorphism of the UGT2B17 gene is associated with increased risk for prostate cancer and correlated to gene expression in the prostate

Metabolism of androgens includes glucuronidation, the major pathway of steroid elimination in several steroid target tissues. Glucuronidation is catalysed by UDP-glucuronosyltransferases (UGTs). UGT2B17 has been shown to be particularly active against androgens and is highly abundant in the prostate. Recently, we discovered that deletion of the UGT2B17 gene is associated with low or undetectable urinary testosterone levels. Here, we determined the phenotypic outcome of the deletion by quantifying the UGT2B17 mRNA expression in normal prostate tissues in individuals with different genotypes. Additionally, the frequency of UGT2B17 deletion polymorphism was studied in a Swedish population-based case-control study including 176 patients diagnosed with prostate cancer and 161 controls. We found that the individuals homozygous for the insertion allele expressed 30 times more UGT2B17 mRNA in prostate tissue than the heterozygotes. Carriers of the deletion allele had a significantly increased risk of prostate cancer (OR=2.07; 95% CI=1.32-3.25). In conclusion, these results show the UGT2B17 deletion polymorphism is associated with prostate cancer risk.

Gender differences in renal nuclear receptors and aryl hydrocarbon receptor in 5/6 nephrectomized rats

This study was aimed at delineating molecular pathways essential in gender-different pathogenesis of chronic kidney diseases (CKD). Renal transcripts of nuclear receptors and metabolic enzymes in male and female kidneys from 5/6 nephrectomized (Nx) rats 7 weeks post-Nx were examined using branched DNA signal amplification assay. Nx-males had marked kidney injury coupled with anemia and malnutrition. Nx-females had moderate renal injury, and were free of albuminuria, anemia, and malnutrition. Nx-males had systemic and renal inflammation, which were largely absent in Nx-females. Blood 17beta-estradiol, testosterone, and corticosterone did not change, whereas urinary testosterone decreased in both genders. Compared to males, female kidneys had higher androgen receptor (AR) and aryl hydrocarbon receptor (AhR) but lower estrogen receptor alpha (ERalpha). Compared to Nx-males, female remnant kidneys had less decreases in ERalpha and peroxisome proliferator-activated receptor alpha (PPARalpha), had no induction of AR and decrease of acyl-CoA oxidase, whereas had induction of cytochrome P450 4a1 (Cyp4a1) but decrease of AhR. Renal protein expression of a 52-kDa isoform of Wilm's tumor 1 (WT1), transcription factor critical in nephrogenesis, decreased dramatically in Nx-males but largely preserved in Nx-females. In conclusion, gender divergences in basal expression and alteration of ERalpha, AR, AhR, WT1, and PPARalpha/Cyp4a1 during CKD may explain gender differences in CKD progression and outcome of renal transplantation.

A diurnal variation in testicular hormone production is maintained following gonadotrophin suppression in normal men

A diurnal variation in serum testosterone in adult men is well recognized, but whether this occurs during exogenous testosterone administration and the degree to which it is endogenous to the testis is unclear. A clinical research centre investigation of testicular function in normal men. Twenty normal men were recruited, 10 of whom were investigated during administration of testosterone with etonogestrel to suppress gonadotrophin secretion. Hourly blood samples were taken over 24 h for measurement of testosterone, inhibin B, LH, FSH and cortisol. Urinary excretion of testosterone and the testicular steroid epitestosterone was also measured. In the controls, a diurnal variation in serum testosterone and LH but not FSH was detected. The treated group had similar testosterone concentrations but showed no diurnal variation. Periodicity was also detected in inhibin B concentrations in 5 of the controls and in 9 of the treated group, who also showed synchrony not seen in the controls. Both groups showed diurnal variation in cortisol. Urinary testosterone excretion did not show a diurnal variation in either group, but this was apparent for epitestosterone with a morning peak in both groups despite the markedly lower excretion in the treated men. The diurnal variation of testosterone in normal men is due to a change in secretion rather than in clearance and is largely LH driven. An endogenous rhythm in both testicular steroidogenesis (epitestosterone) and Sertoli cell function (inhibin B) is also present.

Development of an immunosensor for the detection of testosterone in bovine urine

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H 2O 2) substrate system, at +100 mV. The EC 50 values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL −1 and 960 pg mL −1, respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL −1 to 40 ng mL −1; while that in urine ranged from 0.03 ng mL −1 to 1.6 ng mL −1. The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL −1 and 1.8 pg mL −1. Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV < 13%, n = 3) and repeatability (CV < 9%, n = 3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 °C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.

A recombinant Fab fragment-based electrochemical immunosensor for the determination of testosterone in bovine urine

This work describes the development of an electrochemical, recombinant Fab fragment-based immunosensor for the detection of testosterone in bovine urine. The sensor comprised of a testosterone conjugate on the surface of screen-printed electrodes, and recognition followed by an anti-testosterone Fab fragment. The use of an IgG-horseradish peroxidase conjugate determined the degree of competition. Chronoamperometry at a potential of +100 mV, was chosen to reductively measure the product of the catalysis of 3,3',5,5'-tetramethylbenzidine catalysis. ELISA was primarily used to investigate the assay system, prior to transferring to SPEs. The final Fab-based sensor exhibited the linear range of 300-40,000 pg/ml with limit of detection of 90+/-13 pg/ml. Furthermore, the developed Fab sensor allowed for the determination of testosterone in bovine urine directly after dilution, omitting the necessity of extraction and hydrolysis. Comparison of administrated bovine urine samples between the developed Fab sensor and GC-MS data showed quantitative or semi-quantitative results and enabled identification of suspicious samples for further extensive analysis by established analytical techniques. With simple sample preparation, low limit of detection, and good repeatability, the proposed method can offer alternative advantages as a primary screening tool for meat quality control.

Does timing of androgen deprivation influence radiation-induced toxicity? A secondary analysis of Radiation Therapy Oncology Group protocol 9413

4655 Background: We conducted a secondary analysis of RTOG 9413 to compare if the timing of antiandrogen-therapy, concomitant versus adjuvant, influences the incidence of rectal toxicity in whole pelvic radiotherapy. Methods: For the purpose of this secondary analysis, we analyzed the 2 of the 4 arms of the study, in which all patients received radiotherapy to the whole pelvis followed by a boost to the prostate and excluded the two arms that received prostate only radiotherapy. The 2 arms differed only in the timing of the total of 4 months of total androgen deprivation (TAD): arm I (320 patients), TAD was begun 2 months before the start of radiotherapy and continued during radiotherapy. Arm III (319 patients), TAD started immediately after the completion of radiotherapy. Both acute rectal and acute urinary toxicities (CTC v.2.0), testosterone (measured at baseline and yearly after) and other patients data were modeled using the multivariate logistic regression and the multivariate Cox-proportional hazards regression. Results: Median follow up for all patients is 6.0 and 5.8 years (arm I and II, resp.). 43 (13%) patients in each arm had abnormally low testosterone before start of TAD. Late grade 2 - 5 rectal toxicity occurred in 16% and 13% and urinary toxicity in 18% and 20% (arm I and II, resp.). Frequency (or occurrence) of late rectal toxicity (grade 0–1 vs. 2–5, p = 0.2170) and late urinary toxicity (grade 0–1 vs. 2–5, p = 0.4204) are not significantly different between the two arms. The only risk factors for late rectal toxicity in a multivariate regression model was acute rectal toxicity (OR 1.48, p = 0.025), but not abnormal testosterone level at baseline (p = 0.718) or treatment arm (p = 0.874). For late urinary toxicity: age (OR = 1.588, p = 0.010), RT field size (OR 1.004, p &lt; 0.025), baseline testosterone (OR 1.718, p = 0.028), and acute (grade 2–4) toxicity (OR = 1.664, p = 0.006) but not treatment arm (p = 0.928) were risk factors. Conclusions: While late toxicity was not different for concomitant vs. adjuvant hormonal therapy, an abnormally low testosterone level at baseline is a risk factor for late urinary toxicity and not late rectal toxicity. No significant financial relationships to disclose.

Stability studies of testosterone and epitestosterone glucuronides in urine

The stability of testosterone glucuronide (TG), epitestosterone glucuronide (EG) and the T/E ratio in urine has been studied. Samples were analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Urine samples were submitted to a solid-liquid cleanup followed by extraction of unconjugated testosterone (T) and epitestosterone (E) with tert-butyl methyl ether (free fraction). The remaining aqueous phase was hydrolyzed with beta-glucuronidase and extracted at alkaline pH with n-pentane. Analytes were analyzed by GC/MS as their enol-trimethylsilyl (TMS) derivatives. The urine for stability testing was obtained from an excretion study after the administration of T to healthy volunteers. The homogeneity of the sample was verified before starting the stability study. The stability of TG and EG was evaluated at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4 degrees C and -20 degrees C was determined at different time intervals for 22 months. For short-term stability testing, analyte concentration was evaluated in urine stored at 37 degrees C for 3 and 7 days. The effect of repeated freezing (at -20 degrees C) and thawing (at room temperature) was studied for up to three cycles. Data obtained in this work demonstrated the stability of TG, EG and the T/E ratio in sterilized urine samples stored at 4 and -20 degrees C for 22 months and after going through repeated freeze/thaw cycles. Decreases in concentration were observed after 7 days of storage at 37 degrees C due to the partial cleavage of the glucuronide conjugates; however, the T/E ratio was not affected. These results show the feasibility of preparing reference materials containing TG and EG to be used for quality control purposes.

Testosterone in juvenile and adolescent male chimpanzees (Pan troglodytes): Effects of dominance rank, aggression, and behavioral style

Testosterone is a steroid hormone with diverse effects on male reproductive function and behavior. The relationship between testosterone and social behavior such as mating and aggression has been investigated in a variety of primate species, but few such studies have been conducted on chimpanzees, and even fewer on primates during the juvenile and adolescent periods. This study explores the relationship between baseline urinary testosterone and behavioral variables including dominance rank, rates of aggression toward peers, and behavioral style in 16 juvenile and adolescent male chimpanzees (Pan troglodytes) living at the New Iberia Research Center in Louisiana. Behavioral observations and urine collection occurred during four research periods, each a year apart. After correcting for the positive association between testosterone and age, testosterone was positively associated with both dominance rank and rates of aggression directed at others. It was negatively associated with rates of aggression received. Individuals scoring highest in the "mellow" behavioral style component showed higher levels of testosterone than individuals scoring lowest in this component, an effect that may be partially due to the confounding effect of rank. The results of this study suggest that hormonal changes during the period preceding adulthood are not simply programmed physiological processes tied primarily to age-related change, but that important age-independent relationships with behavior also exist.

Longitudinal profiling of urinary steroids by gas chromatography/combustion/isotope ratio mass spectrometry: Diet change may result in carbon isotopic variations

Longitudinal profiling of urinary steroids was investigated by using a gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) method. The carbon isotope ratio of three urinary testosterone (T) metabolites: androsterone, etiocholanolone, 5beta-androstane-3alpha,17beta-diol (5beta-androstanediol) together with 16(5alpha)-androsten-3alpha-ol (androstenol) and 5beta-pregnane-3alpha,20alpha-diol (5beta-pregnanediol) were measured in urine samples collected from three top-level athletes over 2 years. Throughout the study, the subjects were living in Switzerland and were residing every year for a month or two in an African country. (13)C-enrichment larger than 2.5 per thousand was observed for one subject after a 2-month stay in Africa. Our findings reveal that (13)C-enrichment caused by a diet change might be reduced if the stay in Africa was shorter or if the urine sample was not collected within the days after return to Switzerland. The steroids of interest in each sample did not show significant isotopic fractionation that could lead to false positive results in anti-doping testing. In contrast to the results obtained with the carbon isotopic ratio, profiling of urinary testosterone/epitestosterone (T/E) ratios was found to be unaffected by a diet change.