New insights for identification of doping with recombinant human erythropoietin micro-doses after high hydration.

To minimize the chances of being caught after doping with recombinant human erythropoietins (rhEPO), athletes have turned to new practices using micro-doses and excess fluid ingestion to accelerate elimination and decrease the probability of detection. Our objective was to test the sensitivity of detection by validated methods (IEF: isoelectric focusing; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis) when such practices are used. First, after a three-week rhEPO boost period and 10 days of wash out, detection of a single 900 IU micro-dose of Eprex® was evaluated in healthy male subjects. After an injection in the evening, urine and plasma samples were collected the following morning. Half of the subjects then drank a bolus of water and new samples were collected 80 min later. Interestingly, rhEPO was detected in 100% of the samples even after water ingestion. A second similar protocol was then performed with a single injection of a micro-dose of rhEPO (500 IU or 900 IU), without a prior rhEPO boost. In addition, urine and plasma samples were also collected 15 and 20 h post rhEPO administration. Once again drinking water did not affect the rate of detection. Urine appeared a better matrix to detect micro-doses after 10 h, enabling between 92% and 100% of identification at that time. The rate of identification decreased rapidly thereafter, in particular for the 500 IU micro-dose. However IEF analysis still resulted in 71% identification of rhEPO in urine after 20 h. These results could help to define a better strategy for controlling and identifying athletes using rhEPO micro-doses. Copyright © 2016 John Wiley & Sons, Ltd.

Time-course of prednisone effects on hormonal and inflammatory responses at rest and during resistance exercise.

Glucocorticoids are among the most commonly used drugs. They are widely administered for acute and chronic musculoskeletal pain, as well as for several other pain syndromes, although their therapeutic use is sometimes diverted for doping purposes. Their time-course effects on hormonal and inflammatory responses nevertheless remain poorly understood, both at rest and during exercise. We therefore studied the alterations induced by 1 week of prednisone treatment (60 mg daily) in recreationally trained male athletes after 2 days (i. e., acute) and 7 days (i. e., short-term). Hormonal (i. e., DHEA, DHEA-S, aldosterone, and testosterone) and pro- and anti-inflammatory markers (i. e., IL-6, IL-10, and IL-1ß) were investigated at rest and after resistance exercise. A significant decrease in DHEA and DHEA-S (p<0.01) without change in the DHEA/DHEA-S ratio, aldosterone, or testosterone was demonstrated after acute prednisone intake. A significant increment in IL-10 and a significant decrement in IL-6 (p<0.05) were also observed with prednisone both at rest and during exercise, without significant change in IL-1ß. Continued prednisone treatment led to another significant decrease in both DHEA and DHEA-S (p<0.05), whereas no change in the inflammatory markers was observed between days 2 and 7. Our data demonstrate that the anti-inflammatory effects of prednisone were maximal and stable from the beginning of treatment, both in rest and exercise conditions. However, hormonal concentrations continued to decline during short-term intake. Further studies are needed to determine the effects of hormonal time-course alterations with longer glucocorticoid treatment and the clinical consequences.

Oral contraceptive use and saliva diurnal pattern of metabolic steroid hormones in young healthy women.

BACKGROUND: The impact of oral contraceptives (OCs) on the saliva diurnal pattern of metabolic steroid hormones remained unknown. STUDY DESIGN: Saliva samples were taken from young healthy women (11 OC users, 10 non-OC users) to analyze cortisol, dehydroepiandrosterone (DHEA) and testosterone 4 times (days 1, 8, 15 and 22) over one menstrual cycle. RESULTS: OC use decreased saliva testosterone concentrations (p<.01) under all conditions of day and time, but not saliva cortisol. OC also decreased saliva DHEA concentrations during the first part of the day (p<.05), with a dampened amplitude in its diurnal pattern. CONCLUSION: The clinical relevance requires further study.

DHEA, physical exercise and doping.

The dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) concentrations during acute and chronic exercise (training) have been investigated only fairly recently. DHEA is generally preferred to DHEA-S for exploring the acute exercise repercussions in laboratory or field tests because of its shorter elimination half-life. Conversely, DHEA-S is preferred to estimate chronic adaptations. Both can be measured noninvasively in saliva, and it is therefore possible to follow these hormone responses in elite athletes during competitive events and in healthy and pathological populations, without imposing additional stress. Indeed, the correlation between saliva and serum concentrations is high for steroid hormones, both at rest and during exercise. In this review, we will first summarize the current knowledge on the DHEA/DHEA-S responses to exercise and examine the potential modulating factors: exercise intensity, gender, age, and training. We will then discuss the ergogenic effects that athletes expect from the exogenous administration of DHEA and the antidoping methods of analysis currently used to detect this abuse.

Therapeutic glucocorticoid administration alters the diurnal pattern of dehydroepiandrosterone.

Significant alteration in hypothalamic-pituitary-adrenal function has been demonstrated in patients after short-term glucocorticoid therapy, but its impact on the circadian rhythm of steroid hormones has never been investigated. This study examined the effects of short-term prednisone administration on the diurnal patterns of dehydroepiandrosterone (DHEA) and testosterone. Saliva samples were collected from 11 healthy, physically active, male volunteers for DHEA and testosterone analysis, as follows: every 4 h from 0800 to 2000 h on 2 control days without medication, and after 1 week of oral therapeutic prednisone treatment (60 mg daily) (days 0-3). Overall, a diurnal decline in the two steroid hormones was observed on the control days. After short-term glucocorticoid administration, DHEA concentrations were significantly decreased with a complete disappearance of the DHEA diurnal pattern, which lasted 2 days post-treatment. No glucocorticoid effect was observed for testosterone. The results indicate that short-term prednisone treatment affects the circadian pattern of saliva DHEA but not testosterone in healthy active volunteers. Further studies are necessary to determine whether this alteration in DHEA circadian pattern has clinical consequences in patients with chronic glucocorticoid therapy.

The diurnal patterns of cortisol and dehydroepiandrosterone in relation to intense aerobic exercise in recreationally trained soccer players.

Diurnal patterns of cortisol and dehydroepiandrosterone (DHEA) secretion, the two main peripheral secretory products of the hypothalamic-pituitary-adrenal neuroendocrine stress axis, have been well characterized in rest conditions but not in relation to physical exercise. The purpose of this investigation was therefore to determine the effects of an intense 90-min aerobic exercise on the waking diurnal cortisol and DHEA cycles on three separate days [without exercise, with morning exercise (10:00-11:30 h), and with afternoon exercise (14:00-15:30 h)] in nine recreationally trained soccer players. Saliva samples were collected at awakening, 30 min after awakening, and then every 2 h from 08:00 to 22:00 h. A burst of secretory activity was found for cortisol (p < 0.01) but not for DHEA after awakening. Overall, diurnal decline for both adrenal steroids was observed on resting and exercise days under all conditions. However, there was a significant increase in salivary cortisol concentrations on the morning-exercise and afternoon-exercise days at, respectively, 12:00 h (p < 0.05) and 16:00 h (p < 0.01), versus the other trials. This acute response to exercise was not evident for DHEA. The results of this investigation indicate that 90 min of intense aerobic exercise does not affect the circadian pattern of salivary adrenal steroids in recreationally trained athletes over a 16-h waking period, despite a transitory increase in post-exercise cortisol concentration. Further studies are necessary to determine whether these results are applicable to elite athletes or patients with cortisol or DHEA deficiency.

Short-term glucocorticoid intake and metabolic responses during long-lasting exercise.

The aim of the study was to evaluate the effects of short-term glucocorticoid treatment on plasma amino acids, free fatty acids, blood glucose, and several hormones in healthy volunteers performing long-lasting exercise. 9 young female subjects exercised 2 h at 50-55% VO2 max twice, once after placebo (Pla) ingestion and once after prednisone (Cor, 50 mg/day/7 days) ingestion, according to a double-blind and randomized protocol. Blood samples were tat rest and during exercise for measurement of amino acids, free fatty acids, blood glucose, adrenocorticotrophic hormone (ACTH), growth hormone, dehydroepiandrosterone (DHEA), insulin, and glucagon. Both ACTH and DHEA values were significantly decreased by Cor vs. Pla (p < 0.01) throughout exercise, and Cor intake also induced lower growth hormone concentrations vs. Pla (p < 0.05) from 60 min to the end of exercise. No significant difference in glucagon, insulin or free fatty acid values was found between the treatments. Branched-chain amino acids and other essential amino acids were significantly higher after Cor vs. Pla from 60 min to the end of exercise (p < 0.05), whereas blood glucose was significantly higher from 90 min to the end of exercise (p < 0.05). We conclude that short-term glucocorticoid intake induces marked hormonal and metabolic changes during long-lasting exercise. Proteolysis can increase with glucocorticoid during this type of exercise and the related higher plasma amino acid concentrations may contribute as energy substrates. Further studies will be necessary to explore and accurately quantify the mechanisms of proteolysis and glyconeogenesis induced by short-term glucocorticoid intake during this type of exercise.

Correlation between plasma and saliva adrenocortical hormones in response to submaximal exercise.

This study examined the relationships between plasma and saliva adrenocortical hormones in response to long-duration submaximal exercise. In nine healthy, physically active, female volunteers, blood and saliva samples were taken at rest and every 30 min during a 120-min cycling trial at 50-55% VO(2max) for cortisol and dehydroepiandrosterone (DHEA) analysis. Correlation analysis revealed a moderate but significant relationship between plasma and saliva cortisol (r = 0.35, P < 0.02) and plasma and saliva DHEA (r = 0.47, P < 0.001) during the submaximal exercise. When expressed in percent of resting values, the correlations between the plasma and saliva concentrations were higher for both hormones during the exercise (cortisol: r = 0.72; DHEA: r = 0.68, P < 0.001). The results thus suggest that, even under prolonged exercise conditions, non-invasive saliva samples may offer a practical approach to assessing pituitary-adrenal function, especially when compared with individual basal values.

Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures.

"Double-blotting" (DB) was developed to overcome the problem of non-specific binding of secondary antibodies in immunoblotting (IB). After it had been probed by the primary antibody, the membrane with the blotted proteins was assembled with a second blank membrane and submitted to a second blotting under acidic conditions. The primary antibody molecules were thus desorbed from their corresponding antigen and transferred onto the second membrane, whereas the antigen and the interfering proteins remained bound to the first one. The second membrane could then be probed by the secondary antibodies without the risk of non-specific binding. This method was developed for the study of erythropoietin (EPO) in concentrated urine since a strong non-specific binding of biotinylated secondary antibodies to some urinary proteins had been observed using classical IB protocols.

Effects of acute ingestion of salbutamol during submaximal exercise.

To assess the eventual effects of acute oral salbutamol intake on performance and metabolism during submaximal exercise, nine healthy volunteers completed two cycling trials at a power corresponding to 80-85% VO2max, after either placebo (Pla) or salbutamol (Sal, 6 mg) treatment, according to a double-blind randomized protocol. Blood samples were collected both at rest and during exercise (5 min-, 10 min-, 15 min-exhaustion) for C-peptide, FFA, lactate and blood glucose measurements. Cycling performance was significantly improved in the Sal vs. Pla trials (p < 0.05). After Sal intake, resting C-peptide, lactate, FFA and blood glucose values were higher whereas exercise lactate and free fatty acid concentrations were greater during and at the conclusion of the exercise period (p < 0.05). These results suggest that acute salbutamol ingestion improved performance during submaximal exercise probably through an enhancement of the overall contribution to energy production from both aerobic and anaerobic metabolisms.

Effects of short-term oral salbutamol administration on exercise endurance and metabolism.

The present study examined whether oral short-term administration of salbutamol (Sal) modifies performance and selected hormonal and metabolic variables during submaximal exercise. Eight recreational male athletes completed two cycling trials at 80-85% peak O(2) consumption until exhaustion after either gelatin placebo (Pla) or oral Sal (12 mg/day for 3 wk) treatment, according to a double-blind and randomized protocol. Blood samples were collected at rest, after 5, 10, and 15 min, and at exhaustion to determine growth hormone (GH), cortisol, testosterone, triiodothyronine (T(3)), C peptide, free fatty acid (FFA), blood glucose, lactate, and blood urea values. Time of cycling was significantly increased after chronic Sal intake (Sal: 30.5 +/- 3.1 vs. Pla: 23.7 +/- 1.6 min, P < 0.05). No change in any variable was found before cycling except a decrease in blood urea concentration and an increase in T(3) after Sal that remained significant throughout the exercise test (P < 0.05). Compared with rest, exercise resulted in a significant increase in GH, cortisol, testosterone, T(3), FFAs, and lactate and a decrease in C peptide after both treatments with higher exercise FFA levels and exhaustion GH concentrations after Sal (P < 0.05). Sal but not Pla significantly decreased exercise blood glucose levels. From these data, short-term Sal intake did appear to improve performance during intense submaximal exercise with concomitant increase in substrate availability and utilization, but the exact mechanisms involved need further investigation.

[Evaluation of the cytotoxicity of antiseptics used in current practice on cultures of fibroblasts and keratinocytes]. / Evaluation de la cytotoxicité des antiseptiques utilisés en pratique courante sur des cultures de fibroblastes et de kératinocytes.

Infection is the greatest problem in burn patients and topical antiseptics must be chosen with great care especially when cultured skin is grafted. We examined the cytotoxicity of 6 antiseptics commonly used on cultured human fibroblasts and keratinocytes. Cultured cells were exposed for 15 min to Hibitane (chlorhexidine), Biseptine (chlorhexidine + benzalkonium chloride + benzylic alcool), dermic Betadine (polvidone iodine + nonoxinol), scrub Betadine (polyvidone iodine + quaternary ammonium) and gynecologic Betadine (polyvidone iodine). The cell viability was determined using the MTT test. At therapeutic concentration all the antiseptics were cytotoxic for fibroblasts and keratinocytes. The data suggest that the antiseptics must be used in function of the time of the grafting of the cultured epithelium.

Cytotoxicity evaluation of antiseptics and antibiotics on cultured human fibroblasts and keratinocytes.

Infection is the greatest problem in burn patients and topical antimicrobial agents must be chosen with great care, especially when cultured skin is grafted. We examined the cytotoxic effect of six antiseptics and six antibiotics commonly used on cultured human fibroblasts and keratinocytes. Cultured cells were exposed for 15 min to Hibitane (chlorhexidine), Biseptine (chlorhexidine+benzalkonium chloride+benzylic alcohol), Benzalkonium Chloride, Yellow Betadine (polyvidone-iodine+nonoxinol), Betadine Scrub (polyvidone-iodine+quaternary ammonium) and Green Betadine (polyvidone-iodine) and viability was determined using the MTT test. At therapeutic concentrations all the antiseptics are cytotoxic for fibroblasts and keratinocytes. Additionally the cells were exposed for 48 h to vancomycin, colistin, amikacin, imipeneme, pefloxaxin, piperacillin and cell viability was determined using the MTT test. The concentrations of antibiotics corresponding to the plasma peak obtained after therapeutic application were not cytotoxic to the tested cells. The CD50 was much higher than the MIC (from 125 to 875 times for keratinocytes and from 1400 to 5900 times for fibroblasts). These data suggest that commonly applied antiseptics must not be used before grafting cultured skin grafts. After grafting any infection can be controlled with topical applications of appropriate antibiotics.

Radioimmunofixation of human ferritin following serum isoelectric focusing.

Radioimmunofixation of human ferritin following isoelectric focusing of serum was developed to study the microheterogeneity of this protein in native serum without previous purification or concentration. This method requires only 2-10 microliter of serum and can be used with levels of ferritin as low as 10 micrograms/l. In this way, the extensive microheterogeneity of this protein was revealed, since in some cases it produced as many as 35 bands with isoelectric points in a pH range of 4.95-5.9. Very different isoelectric focusing patterns (spectrotypes) of ferritin were observed during the investigation of pathological sera. The high sensitivity of this technique makes it useful for the investigation of serum ferritin in diseases involving modifications of the metabolism of this protein.

A fast silver staining method for protein detection after isoelectric focusing in agarose gels.

A sensitive staining method was developed for detecting proteins in agarose gels after isoelectric focusing. Its sensitivity is about 20 times that of the Coomassie blue R-250 staining technique, and the time required is only 10 min.

[Fibrotic interstitial pneumopathies. Collagenolytic activity of the alveolar fluid]. / Pneumopathies interstitielles fibrosantes. Activité collagénolytique du liquide alvéolaire.

Collagenolytic enzyme release in alveolar structures is probably one of the initial events leading to impaired balance between collagen synthesis and degradation in the connective matrix of the lung, resulting in pulmonary fibrosis. The collagenolytic activity was determined in the bronchoalveolar fluid of 40 normal subjects or patients with miscellaneous pulmonary diseases and was found to be present in seventeen, viz.: 7/7 patients with interstitial fibrosis, irrespective of its origin: 4/4 patients with radiation pneumonitis; 4/15 patients with sarcoidosis and 2/2 patients with transient eosinophilic pneumopathy. There was no evidence of fibrosis in the 23 patients who showed no collagenolytic activity. Thus, collagenolytic enzymes are present in the alveolar structures of patients with interstitial pulmonary diseases of diverse origin capable of leading to fibrosis. Monitoring the release of this enzyme by bronchoalveolar lavage could be useful to evaluate the risk of fibrosis in such patients.

Role of sialic acid in the microheterogeneity of serum thyroxine-binding globulin. Study by two-dimensional isoelectric focusing.

A new technique combining a neuraminidase treatment of thyroxine-binding globulin after serum isoelectric focusing and a second-dimensional isoelectric focusing was developed to study the role of sialic acid in the microheterogeneity of native thyroxine-binding globulin. By showing the change of PI occasioned by the desialylation for each of the bands constituting the thyroxine-binding globulin pattern separately, this procedure clearly demonstrated that the microheterogeneity of the native protein could not be imputed to the varying sialic acid content of the bands only. We suggest that at least three molecules of thyroxine-binding globulin, with probably slight differences in their amino acid composition, are present in the serum, and that different degrees of sialylation ensure greater microheterogeneity of this protein.

Microhetero;geneity and polymorphism of human serum thryroxine-binding globulin. Study by isoelectric focusing and radioprint immunofixation.

A new technique of radioprint immunofixation following isoelectric focusing was applied to the study of microheterogeneity of thyroxine-binding globulin without purification, in individual sera; this protein appeared to give rise to at least seven bands with isoelectric points in the range of pI 4.2--5.0. Furthermore, two different distributions of the intensity of these bands were found in the sera investigated, suggesting the existence of thyroxine-binding globulin polymorphism in man.