Endogenous steroid profiling in the athlete biological passport.

The Athlete Biological Passport (ABP) is an individual electronic document that collects data regarding a specific athlete that is useful in differentiating between natural physiologic variations of selected biomarkers and deviations caused by artificial manipulations. A subsidiary of the endocrine module of the ABP, that which here is called Athlete Steroidal Passport (ASP), collects data on markers of an altered metabolism of endogenous steroidal hormones measured in urine samples. The ASP aims to identify not only doping with anabolic-androgenic steroids, but also most indirect steroid doping strategies such as doping with estrogen receptor antagonists and aromatase inhibitors. Development of specific markers of steroid doping, use of the athlete's previous measurements to define individual limits, with the athlete becoming his or her own reference, the inclusion of heterogeneous factors such as the UDPglucuronosyltransferase B17 genotype of the athlete, the knowledge of potentially confounding effects such as heavy alcohol consumption, the development of an external quality control system to control analytical uncertainty, and finally the use of Bayesian inferential methods to evaluate the value of indirect evidence have made the ASP a valuable alternative to deter steroid doping in elite sports. The ASP can be used to target athletes for gas chromatography/combustion/ isotope ratio mass spectrometry (GC/C/IRMS) testing, to withdraw temporarily the athlete from competing when an abnormality has been detected, and ultimately to lead to an antidoping infraction if that abnormality cannot be explained by a medical condition. Although the ASP has been developed primarily to ensure fairness in elite sports, its application in endocrinology for clinical purposes is straightforward in an evidence-based medicine paradigm.

Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that can be activated by various xenobiotics and natural fatty acids. These transcription factors primarily regulate genes involved in lipid metabolism and also play a role in adipocyte differentiation. We present the expression patterns of the PPAR subtypes in the adult rat, determined by in situ hybridization using specific probes for PPAR-alpha, -beta and -gamma, and by immunohistochemistry using a polyclonal antibody that recognizes the three rat PPAR subtypes. In numerous cell types from either ectodermal, mesodermal, or endodermal origin, PPARs are coexpressed, with relative levels varying between them from one cell type to the other. PPAR-alpha is highly expressed in hepatocytes, cardiomyocytes, enterocytes, and the proximal tubule cells of kidney. PPAR-beta is expressed ubiquitously and often at higher levels than PPAR-alpha and -gamma. PPAR-gamma is expressed predominantly in adipose tissue and the immune system. Our results suggest new potential directions to investigate the functions of the different PPAR subtypes.

Effect of somatostatin on duodenal glucose absorption in man.

OBJECTIVE: The hyperglycemic hyperinsulinemic clamp technique using intraduodenally infused glucose is an attractive tool for studying postprandial glucose metabolism under strictly controlled conditions. Because it requires the use of somatostatin (SST), we examined, in this study, the effect of SST on intestinal glucose absorption. CONTEXT: Twenty-six normal volunteers were given a constant 3-h intraduodenal infusion of glucose (6 mg.kg(-1).min(-1)) labeled with [2-(3)H]glucose for glucose absorption measurement. During glucose infusion, 19 subjects received iv SST at doses of 10-100 ng.kg(-1).min(-1) plus insulin and glucagon, and seven subjects were studied under control conditions. In the controls, glucose was absorbed at a rate that, after a 20-min lag period, equaled the infusion rate. RESULTS: With all the doses of SST tested, absorption was considerably delayed but equaled the rate of infusion after 3 h. At that time, only 5 +/- 2% of the total amount of infused glucose was unabsorbed in the control subjects vs. 36 +/- 2% (P < 0.001) in the SST-infused subjects. In the latter, the intraluminal residue was almost totally absorbed within 40 min of the cessation of SST infusion. At the lowest dose of SST tested (10 ng.kg(-1).min(-1)), suppression of insulin secretion was incomplete. CONCLUSION: These properties of SST hamper the use of intraduodenal hyperglycemic hyperinsulinemic clamps as a tool for exploring postprandial glucose metabolism.

Interaction between calcium intake and menarcheal age on bone mass gain: an eight-year follow-up study from prepuberty to postmenarche.

Both late menarcheal age and low calcium intake (Ca intake) during growth are risk factors for osteoporosis, probably by impairing peak bone mass. We investigated whether lasting gain in areal bone mineral density (aBMD) in response to increased Ca intake varies according to menarcheal age and, conversely, whether Ca intake could influence menarcheal age. In an initial study, 144 prepubertal girls were randomized in a double-blind controlled trial to receive either a Ca supplement (Ca-suppl.) of 850 mg/d or placebo from age 7.9-8.9 yr. Mean aBMD gain determined by dual energy x-ray absorptiometry at six sites (radius metaphysis, radius diaphysis, femoral neck, trochanter, femoral diaphysis, and L2-L4) was significantly (P = 0.004) greater in the Ca-suppl. than in the placebo group (27 vs. 21 mg/cm(2)). In 122 girls followed up, menarcheal age was recorded, and aBMD was determined at 16.4 yr of age. Menarcheal age was lower in the Ca-suppl. than in the placebo group (P = 0.048). Menarcheal age and Ca intake were negatively correlated (r = -0.35; P < 0.001), as were aBMD gains from age 7.9-16.4 yr and menarcheal age at all skeletal sites (range: r = -0.41 to r = -0.22; P < 0.001 to P = 0.016). The positive effect of Ca-suppl. on the mean aBMD gain from baseline remained significantly greater in girls below, but not in those above, the median of menarcheal age (13.0 yr). Early menarcheal age (12.1 +/- 0.5 yr): placebo, 286 +/- 36 mg/cm(2); Ca-suppl., 317 +/- 46 (P = 0.009); late menarcheal age (13.9 +/- 0.5 yr): placebo, 284 +/- 58; Ca-suppl., 276 +/- 50 (P > 0.05). The level of Ca intake during prepuberty may influence the timing of menarche, which, in turn, could influence long-term bone mass gain in response to Ca supplementation. Thus, both determinants of early menarcheal age and high Ca intake may positively interact on bone mineral mass accrual.

Identification of two steroid-responsive promoters of different strength controlled by the same estrogen-responsive element in the 5'-end region of the Xenopus laevis vitellogenin gene A1.

A structural and functional analysis of the 5'-end region of the Xenopus laevis vitellogenin gene A1 revealed two transcription initiation sites located 1.8 kilobases apart. A RNA polymerase II binding assay indicates that both promoters form initiation complexes efficiently. In vitro, using a transcription assay derived from a HeLa whole-cell extract, the upstream promoter is more than 10-fold stronger than the downstream one. In contrast, both promoters have a similar strength in a HeLa nuclear extract. In vivo, that is in estrogen-stimulated hepatocytes, it is the downstream promoter homologous to the one used by the other members of the vitellogenin gene family, which is 50-fold stronger than the upstream promoter. Thus, if functional vitellogenin mRNA results from this latter activity, it would contribute less than 1% to the synthesis of vitellogenin by fully induced Xenopus hepatocytes expressing the four vitellogenin genes. In contrast, both gene A1 promoters are silent in uninduced hepatocytes. Transfection experiments using the Xenopus cell line B3.2 in which estrogen-responsiveness has been introduced reveal that the strong downstream promoter is controlled by an estrogen responsive element (ERE) located 330 bp upstream of it. The upstream promoter can also be controlled by the same ERE. Since the region comprising the upstream promoter is flanked by a 200 base pair long inverted repeat with stretches of homology to other regions of the X. laevis genome, we speculate that it might have been inserted upstream of the vitellogenin gene A1 by a recombination event and consequently brought under control of the ERE lying 1.5 kilobases downstream.

Techniques for analytical testing of unconventional samples.

Forensic scientists have long detected the presence of drugs and their metabolites in biological materials using body fluids such as urine, blood and/or other biological liquids or tissues. For doping analysis, only urine has so far been collected. In recent years, remarkable advances in sensitive analytical techniques have encouraged the analysis of drugs in unconventional biological samples such as hair, saliva and sweat. These samples are easily collected, although drug levels are often lower than the corresponding levels in urine or blood. This chapter reviews recent studies in the detection of doping agents in hair, saliva and sweat. Sampling, analytical procedures and interpretation of the results are discussed in comparison with those obtained from urine and blood samples.

Novel testis germ cell-specific transcript of the CREB gene contains an alternatively spliced exon with multiple in-frame stop codons.

The gene encoding the cAMP-responsive transcription factor CREB consists of multiple small exons some of which undergo alternative RNA splicing. We describe the finding of a novel transcript of the CREB gene expressed at high levels in the germ cells of the rat testis. The transcript contains an alternatively spliced exon inserted within the sequence encoding the transcriptional transactivation domain of CREB and this exon contains multiple in-frame stop codons. Furthermore, the exon is conserved in both rat and human genes (75% nucleotide identity). Although the function(s) of this RNA or the truncated CREB protein predicted to result from the translation of this unusual transcript is unknown, the high level of expression in the testicular germ cells and remarkable conservation of sequences in rat and human suggests that it may have a unique biological function in these cells.

Monoamine oxidase A down-regulation contributes to high metanephrine concentration in pheochromocytoma.

CONTEXT: The high diagnostic performance of plasma-free metanephrines (metanephrine and normetanephrine) (MN) for pheochromocytoma (PHEO) results from the tumoral expression of catechol-O-methyltransferase (COMT), the enzyme involved in O-methylation of catecholamines (CAT). Intriguingly, metanephrine, in contrast to epinephrine, is not remarkably secreted during a stress in hypertensive or normotensive subjects, whereas in PHEO patients CAT and MN are both raised to high levels. Because epinephrine and metanephrine are almost exclusively produced by the adrenal medulla, this suggests distinct CAT metabolism in chromaffin cells and pheochromocytes. OBJECTIVE: The objective of the study was to compare CAT metabolism between adrenal medulla and PHEO tissue regarding related enzyme expression including monoamine oxidases (MAO) and COMT. DESIGN: A multicenter comparative study was conducted. STUDY PARTICIPANTS: The study included 21 patients with a histologically confirmed PHEO and eight adrenal glands as control. MAIN OUTCOME MEASURES: CAT, dihydroxyphenol-glycol, 3,4-dihydroxyphenylacetic acid, and MN were measured in adrenal medulla and PHEO tissue. Western blot, quantitative RT-PCR and immunofluorescence studies for MAOA, MAOB, tyrosine hydroxylase, dopamine β-hydroxylase, L-amino acid decarboxylase, and COMT were applied on tissue homogenates and cell preparations. RESULTS: At both the protein and mRNA levels, MAOA and COMT are detected less often in PHEO compared with adrenal medulla, conversely to tyrosine hydroxylase, L-amino acid decarboxylase, and dopamine β-hydroxylase, much more expressed in tumor tissue. MAOB protein is detected less often in tumor but not differently expressed at the mRNA level. Dihydroxyphenol-glycol is virtually absent from tumor, whereas MN, produced by COMT, rises to 4.6-fold compared with adrenal medulla tissue. MAOA down-regulation was observed in 100% of tumors studied, irrespectively of genetic alteration identified; on the other hand, MAOA was strongly expressed in all adrenal medulla collected independently of age, gender, or late sympathetic activation of the deceased donor. CONCLUSION: High concentrations of MN in tumor do not only arise from CAT overproduction but also from low MAOA expression, resulting in higher substrate availability for COMT.

Effects of corticosterone pellets on baseline and stress-induced corticosterone and corticosteroid-binding-globulin.

Exogenous administration of glucocorticoids is a widely used and efficient tool to investigate the effects of elevated concentrations of these hormones in field studies. Because the effects of corticosterone are dose and duration-dependent, the exact course of plasma corticosterone levels after exogenous administration needs to be known. We tested the performance of self-degradable corticosterone pellets (implanted under the skin) in elevating plasma corticosterone levels. We monitored baseline (sampled within 3min after capture) total corticosterone levels and investigated potential interactions with corticosteroid-binding-globulin (CBG) capacity and the endogenous corticosterone response to handling in Eurasian kestrel Falco tinnunculus and barn owl Tyto alba nestlings. Corticosterone pellets designed for a 7-day-release in rodents elevated circulating baseline total corticosterone during only 2-3 days compared to placebo-nestlings. Highest levels occurred 1-2days after implantation and levels decreased strongly thereafter. CBG capacity was also increased, resulting in a smaller, but still significant, increase in baseline free corticosterone levels. The release of endogenous corticosterone as a response to handling was strong in placebo-nestlings, but absent 2 and 8 days after corticosterone pellet implantation. This indicates a potential shut-down of the hypothalamo-pituitary-adrenal axis after the 2-3 days of elevated baseline corticosterone levels. 20 days after pellet implantation, the endogenous corticosterone response to handling of nestlings implanted with corticosterone pellets attained similar levels as in placebo-nestlings. Self-degradable pellets proved to be an efficient tool to artificially elevate circulating baseline corticosterone especially in field studies, requiring only one intervention. The resulting peak-like elevation of circulating corticosterone, the concomitant elevation of CBG capacity, and the absence of an endogenous corticosterone response to an acute stressor have to be taken into account.

Whole body protein metabolism and resting energy expenditure in pregnant Gambian women.

Whole body protein metabolism and resting energy expenditure (REE) were measured at 11, 23, and 33 wk of pregnancy in nine pregnant (not malnourished) Gambian women and in eight matched nonpregnant nonlactating (NPNL) matched controls. Rates of whole body nitrogen flux, protein synthesis, and protein breakdown were determined in the fed state from the level of isotope enrichment of urinary urea and ammonia during a period of 9 h after a single oral dose of [15N]glycine. At regular intervals, REE was measured by indirect calorimetry (hood system). Based on the arithmetic end-product average of values obtained with urea and ammonia, a significant increase in whole body protein synthesis was observed during the second trimester (5.8 +/- 0.4 g.kg-1.day-1) relative to values obtained both for the NPNL controls (4.5 +/- 0.3 g.kg-1.day-1) and those during the first trimester (4.7 +/- 0.3 g.kg-1.day-1). There was a significant rise in REE during the third trimester both in the preprandial and postprandial states. No correlation was found between REE after meal ingestion and the rate of whole body protein synthesis.

Abrupt decrease in serum testosterone levels after an oral glucose load in men: implications for screening for hypogonadism.

OBJECTIVE: This study examines the physiological impact of a glucose load on serum testosterone (T) levels in men with varying glucose tolerance (GT). DESIGN: Cross-sectional study. PATIENTS AND METHODS: 74 men (19-74 years, mean 51·4 ± 1·4 years) underwent a standard 75-g oral glucose tolerance test with blood sampling at 0, 30, 60, 90 and 120 min. Fasting serum glucose, insulin, total T (and calculated free T), LH, SHBG, leptin and cortisol were measured. RESULTS: 57% of the men had normal GT, 30% had impaired GT and 13% had newly diagnosed type 2 diabetes. Glucose ingestion was associated with a 25% decrease in mean T levels (delta = -4·2 ± 0·3 nm, P < 0·0001). T levels remained suppressed at 120 min compared with baseline (13·7 ± 0·6 vs 16·5 ± 0·7 nm, P < 0·0001) and did not differ across GT or BMI. Of the 66 men with normal T levels at baseline, 10 (15%) had levels that decreased to the hypogonadal range (<9·7 nm) at one or more time points. SHBG, LH and cortisol levels were unchanged. Leptin levels decreased from baseline at all time points (P < 0·0001). CONCLUSIONS: Glucose ingestion induces a significant reduction in total and free T levels in men, which is similar across the spectrum of glucose tolerance. This decrease in T appears to be because of a direct testicular defect, but the absence of compensatory changes in LH suggests an additional central component. Men found to have low nonfasting T levels should be re-evaluated in the fasting state.

Association between circulating cytokine levels, diabetes and insulin resistance in a population-based sample (CoLaus study).

OBJECTIVE: The associations between inflammation, diabetes and insulin resistance remain controversial. Hence, we assessed the associations between diabetes, insulin resistance (using HOMA-IR) and metabolic syndrome with the inflammatory markers high-sensitive C-reactive protein (hs-CRP), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α). DESIGN: Cross-sectional study. PARTICIPANTS: Two thousand eight hundred and eighty-four men and 3201 women, aged 35-75, participated in this study. METHODS: C-reactive protein was assessed by immunoassay and cytokines by multiplexed flow cytometric assay. In a subgroup of 532 participants, an oral glucose tolerance test (OGTT) was performed to screen for impaired glucose tolerance (IGT). RESULTS: IL-6, TNF-α and hs-CRP were significantly and positively correlated with fasting plasma glucose (FPG), insulin and HOMA-IR. Participants with diabetes had higher IL-6, TNF-α and hs-CRP levels than participants without diabetes; this difference persisted for hs-CRP after multivariate adjustment. Participants with metabolic syndrome had increased IL-6, TNF-α and hs-CRP levels; these differences persisted after multivariate adjustment. Participants in the highest quartile of HOMA-IR had increased IL-6, TNF-α and hs-CRP levels; these differences persisted for TNF-α and hs-CRP after multivariate adjustment. No association was found between IL-1β levels and all diabetes and insulin resistance markers studied. Finally, participants with IGT had higher hs-CRP levels than participants with a normal OGTT, but this difference disappeared after controlling for body mass index (BMI). CONCLUSION: We found that subjects with diabetes, metabolic syndrome and increased insulin resistance had increased levels of IL6, TNF-α and hs-CRP, while no association was found with IL-1β. The increased inflammatory state of subjects with IGT is partially explained by increased BMI.

Expanding the phenotype and genotype of female GnRH deficiency.

Context: GnRH deficiency is a rare genetic disorder of absent or partial pubertal development. The clinical and genetic characteristics of GnRH-deficient women have not been well-described. Objective: To determine the phenotypic and genotypic spectrum of a large series of GnRH-deficient women. Design, Setting, and Subjects: Retrospective study of 248 females with GnRH deficiency evaluated at an academic medical center between 1980 and 2010. Main Outcome Measures: Clinical presentation, baseline endogenous GnRH secretory activity, and DNA sequence variants in 11 genes associated with GnRH deficiency. Results: Eighty-eight percent had undergone pubarche, 51% had spontaneous thelarche, and 10% had 1-2 menses. Women with spontaneous thelarche were more likely to demonstrate normal pubarche (P = 0.04). In 27% of women, neuroendocrine studies demonstrated evidence of some endogenous GnRH secretory activity. Thirty-six percent (a large excess relative to controls) harbored a rare sequence variant in a gene associated with GnRH deficiency (87% heterozygous and 13% biallelic), with variants in FGFR1 (15%), GNRHR (6.6%), and PROKR2 (6.6%) being most prevalent. One woman had a biallelic variant in the X-linked gene, KAL1, and nine women had heterozygous variants. Conclusions: The clinical presentation of female GnRH deficiency varies from primary amenorrhea and absence of any secondary sexual characteristics to spontaneous breast development and occasional menses. In this cohort, rare sequence variants were present in all of the known genes associated with GnRH deficiency, including the novel identification of GnRH-deficient women with KAL1 variants. The pathogenic mechanism through which KAL1 variants disrupt female reproductive development requires further investigation.

Test methods: anabolics.

In the International Olympic Committee (IOC) accredited laboratories, specific methods have been developed to detect anabolic steroids in athletes' urine. The technique of choice to achieve this is gas-chromatography coupled with mass spectrometry (GC-MS). In order to improve the efficiency of anti-doping programmes, the laboratories have defined new analytical strategies. The final sensitivity of the analytical procedure can be improved by choosing new technologies for use in detection, such as tandem mass spectrometry (MS-MS) or high resolution mass spectrometry (HRMS). A better sample preparation using immuno-affinity chromatography (IAC) is also a good tool for improving sensitivity. These techniques are suitable for the detection of synthetic anabolic steroids whose structure is not found naturally in the human body. The more and more evident use, on a large scale, of substances chemically similar to the endogenous steroids obliges both the laboratory and the sports authorities to use the steroid profile of the athlete in comparison with reference ranges from a population or with intraindividual reference values.