RESUMO
Mont Blanc, the summit of Western Europe, is a popular but demanding high-altitude ascent. Drug use is thought to be widespread among climbers attempting this summit, not only to prevent altitude illnesses, but also to boost physical and/or psychological capacities. This practice may be unsafe in this remote alpine environment. However, robust data on medication during the ascent of Mont Blanc are lacking. Individual urine samples from male climbers using urinals in mountain refuges on access routes to Mont Blanc (Goûter and Cosmiques mountain huts) were blindly and anonymously collected using a hidden automatic sampler. Urine samples were screened for a wide range of drugs, including diuretics, glucocorticoids, stimulants, hypnotics and phosphodiesterase 5 (PDE-5) inhibitors. Out of 430 samples analyzed from both huts, 35.8% contained at least one drug. Diuretics (22.7%) and hypnotics (12.9%) were the most frequently detected drugs, while glucocorticoids (3.5%) and stimulants (3.1%) were less commonly detected. None of the samples contained PDE-5 inhibitors. Two substances were predominant: the diuretic acetazolamide (20.6%) and the hypnotic zolpidem (8.4%). Thirty three samples were found positive for at least two substances, the most frequent combination being acetazolamide and a hypnotic (2.1%). Based on a novel sampling technique, we demonstrate that about one third of the urine samples collected from a random sample of male climbers contained one or several drugs, suggesting frequent drug use amongst climbers ascending Mont Blanc. Our data suggest that medication primarily aims at mitigating the symptoms of altitude illnesses, rather than enhancing performance. In this hazardous environment, the relatively high prevalence of hypnotics must be highlighted, since these molecules may alter vigilance.
Assuntos
Ecossistema , Preparações Farmacêuticas/urina , Coleta de Urina/métodos , Automação , Humanos , Masculino , MontanhismoRESUMO
Nonpolar anabolic steroids are doping agents that typically do not provide strong signals by electrospray ionization-mass spectrometry (ESI-MS) owing especially to the low polarity of the functional groups present. We have investigated the addition of anions, in ammonium salt form, to anabolic steroid samples as ionization enhancers and have confirmed that lower instrumental limits of detection (as low as 10 ng/mL for fluoxymesterone-M) are obtained by fluoride anion attachment mass spectrometry, as compared to ESI(+)/(-) or atmospheric pressure photoionization (APPI)(+). Moreover, collision-induced decomposition (CID) spectra of precursor fluoride adducts of the bifunctional steroid "reduced pregnenolone" (containing two hydroxyl groups) and its d4-analogue provide evidence of regiospecific decompositions after attachment of fluoride anion to a specific hydroxyl group of the steroid. This type of charting of specific CID reaction pathways can offer value to selected reaction monitoring experiments (SRM) as it may result in a gain in selectivity in detection as well as in improvements in quantification.
Assuntos
Compostos de Amônio/química , Esteroides/análise , Esteroides/química , Ânions/química , Sais/química , Espectrometria de Massas por Ionização por Electrospray , EstereoisomerismoRESUMO
Chlorazanil (Ordipan, N-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine) is a diuretic agent and as such prohibited in sport according to the regulations of the World Anti-Doping Agency (WADA). Despite its introduction into clinical practice in the late 1950s, the worldwide very first two adverse analytical findings were registered only in 2014, being motive for an in-depth investigation of these cases. Both individuals denied the intake of the drug; however, the athletes did declare the use of the antimalarial prophylactic agent proguanil due to temporary residences in African countries. A structural similarity between chlorazanil and proguanil is given but no direct metabolic relation has been reported in the scientific literature. Moreover, chlorazanil has not been confirmed as a drug impurity of proguanil. Proguanil however is metabolized in humans to N-(4-chlorophenyl)-biguanide, which represents a chemical precursor in the synthesis of chlorazanil. In the presence of formic acid, formaldehyde, or formic acid esters, N-(4-chlorophenyl)-biguanide converts to chlorazanil. In order to probe for potential sources of the chlorazanil detected in the doping control samples, drug formulations containing proguanil and urine samples of individuals using proguanil as antimalarial drug were subjected to liquid chromatography-high resolution/high accuracy mass spectrometry. In addition, in vitro simulations with 4-chlorophenyl-biguanide and respective reactants were conducted in urine and resulting specimens analyzed for the presence of chlorazanil. While no chlorazanil was found in drug formulations, the urine samples of 2 out of 4 proguanil users returned findings for chlorazanil at low ng/mL levels, similar to the adverse analytical findings in the doping control samples. Further, in the presence of formaldehyde, formic acid and related esters, 4-chlorophenyl-biguanide was found to produce chlorazanil in human urine, suggesting that the detection of the obsolete diuretic agent was indeed the result of artefact formation and not of the illicit use of a prohibited substance.
Assuntos
Antimaláricos/metabolismo , Clorobenzenos/urina , Diuréticos/urina , Dopagem Esportivo , Proguanil/metabolismo , Triazinas/urina , Cromatografia Líquida , Humanos , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em TandemRESUMO
Nonspecific interactions between blotted proteins and unrelated secondary antibodies generate false positives in immunoblotting techniques. Some procedures have been developed to reduce this adsorption, but they may work in specific applications and be ineffective in others. "Double-blotting" has been developed to overcome this problem. It consists of interpolating a second blotting step between the usual probings of the blot membrane with the primary antibody and the secondary antibodies. This step, by isolating the primary antibody from the interfering proteins, guarantees the specificity of the probing with the secondary antibody. This method has been developed for the study of erythropoietin in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical immunoblotting protocols. However, its concept makes it usable in other applications that come up against this kind of problem. This method is expected to be especially useful for investigating proteins that are present in minute amounts in complex biological media.
Assuntos
Anticorpos/imunologia , Artefatos , Eritropoetina/análise , Eritropoetina/imunologia , Immunoblotting/métodos , Especificidade de Anticorpos , HumanosRESUMO
As a synthetic analogue of adrenocorticotropic hormone (ACTH), tetracosactide is prohibited in sport by the World Anti-Doping Agency (WADA). An enzyme-linked immunosorbent assay (ELISA) method is proposed for detection of this drug in plasma. Since its structure corresponds to the 24 N-terminal of the 39 amino acids of the natural endogenous peptide ACTH, tetracosactide can be detected with a commercial ELISA kit for ACTH that uses antibodies, the epitopes of which are located in the 1-24 part of ACTH. However, an essential condition for detection specificity is the preliminary total clearance of endogenous ACTH in the plasma samples. This is achieved by a preparative step based on cation-exchange chromatography before ELISA. The method is specific and sensitive (LOD: 30 pg/mL) and may be used as a screening analysis in anti-doping control. The pre-analytical conditions are shown to be of the upmost importance and recommendations for blood collection (EDTA tubes), sample transport (4 °C) and plasma sample storage (-20 °C) are presented.
Assuntos
Cosintropina/análise , Cosintropina/sangue , Ensaio de Imunoadsorção Enzimática , Dopagem Esportivo/prevenção & controle , Humanos , Limite de Detecção , Sensibilidade e EspecificidadeRESUMO
The classic model of tumor suppression implies that malignant transformation requires full "two-hit" inactivation of a tumor-suppressor gene. However, more recent work in mice has led to the proposal of a "continuum" model that involves more fluid concepts such as gene dosage-sensitivity and tissue specificity. Mutations in the tumor-suppressor gene von Hippel-Lindau (VHL) are associated with a complex spectrum of conditions. Homozygotes or compound heterozygotes for the R200W germline mutation in VHL have Chuvash polycythemia, whereas heterozygous carriers are free of disease. Individuals with classic, heterozygous VHL mutations have VHL disease and are at high risk of multiple tumors (e.g., CNS hemangioblastomas, pheochromocytoma, and renal cell carcinoma). We report here an atypical family bearing two VHL gene mutations in cis (R200W and R161Q), together with phenotypic analysis, structural modeling, functional, and transcriptomic studies of these mutants in comparison with classical mutants involved in the different VHL phenotypes. We demonstrate that the complex pattern of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of VHL protein (pVHL) dysfunction in hypoxia signaling pathways. Thus, by studying naturally occurring familial mutations, our work validates in humans the "continuum" model of tumor suppression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Carcinogênese/metabolismo , Mutação , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Carcinoma de Células Renais/genética , Humanos , Neoplasias Renais/genética , Simulação de Dinâmica Molecular , Feocromocitoma/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
All systemically administered glucocorticoids (GC) are prohibited in-competition, because of the potential ergogenic effects. Although short-term GC intake has been shown to improve performance during submaximal exercise, literature on its impact during brief intense exercise appears to be very scant. The purpose of this study was to examine the ergogenic and metabolic effects of prednisone during repeated bouts of high-intensity exercise. In a double-blind randomized protocol, ten recreational male athletes followed two 1-week treatments (Cor: prednisone, 60mg/day or Pla: placebo). At the end of each treatment, they hopped on their dominant leg for 30s three times consecutively and then hopped until exhaustion, with intervals of 5min of passive recovery. Blood and saliva samples were collected at rest and 3min after each exercise bout to determine the lactate, interleukin-6, interleukin-10, TNF-alpha, DHEA and testosterone values. The absolute peak force of the dominant leg was significantly increased by Cor but only during the first 30-s hopping bout (p<0.05), whereas time to exhaustion was not significantly changed after Cor treatment vs Pla (Pla: 119.9±24.7; Cor: 123.1±29.5s). Cor intake lowered basal and end-exercise plasma interleukin-6 and saliva DHEA (p<0.01) and increased interleukin-10 (p<0.01), whereas no significant change was found in blood lactate and TNF-alpha or saliva testosterone between Pla and Cor. According to these data, short-term glucocorticoid intake did not improve endurance performance during repeated bouts of high-intensity exercise, despite the significant initial increase in absolute peak force and anti-inflammatory effect.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Exercício Físico/fisiologia , Glucocorticoides/administração & dosagem , Glucocorticoides/farmacologia , Administração Oral , Adulto , Estudos Cross-Over , Método Duplo-Cego , Teste de Esforço , Glucocorticoides/sangue , Voluntários Saudáveis , Humanos , Masculino , Adulto JovemRESUMO
Metabonomics has become a very valuable tool and many research fields rely on results coming out from this combination of analytical techniques, chemometric strategies, and biological interpretation. Moreover, the matrices are more and more complex and the implications of the results are often of major importance. In this context, the need for pertinent validation strategies comes naturally. The choice of the appropriate chemometric method remains nevertheless a difficult task due to particularities such as: the number of measured variables, the complexity of the matrix and the purposes of the study. Consequently, this paper presents a detailed metabonomic study on human urine with a special emphasis on the importance of assessing the data's quality. It also describes, step by step, the statistical tools currently used and offers a critical view on some of their limits. In this work, 29 urine samples among which 15 samples obtained from tetrahydrocannabinol (delta-9-tetrahydrocannabinol)-consuming athletes, 5 samples provided by volunteers, and 9 samples obtained from athletes were submitted to untargeted analysis by means of ultra high-pressure liquid chromatography-electrospray ionization-time-of-flight mass spectrometry. Next, the quality of the obtained data was assessed and the results were compared to those found in databases. Then, unsupervised (principal component analysis (PCA)) and supervised (ANOVA/PCA, partial least-square-discriminant analysis (PLS-DA), orthogonal PLS-DA) univariate and multivariate statistical methods were applied.
Assuntos
Mineração de Dados/estatística & dados numéricos , Dronabinol/urina , Metabolômica , Cromatografia Líquida de Alta Pressão , Dopagem Esportivo , Dronabinol/metabolismo , Humanos , Análise de Componente Principal , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Competitive swimmers regularly perform apnea series with or without fins as part of their training, but the ergogenic and metabolic repercussions of acute and chronic apnea have not been examined. Therefore, we aimed to investigate the cardiovascular, lactate, arterial oxygen saturation and hormonal responses to acute apnea in relation to performance in male swimmers. According to a randomized protocol, 15 national or regional competitive swimmers were monitored while performing four 100-m freestyle trials, each consisting of four 25-m segments with departure every 30 seconds at maximal speed in the following conditions: with normal frequency breathing with fins (F) and without fins (S) and with complete apnea for the four 25-m segments with (FAp) and without fins (SAp). Heart rate (HR) was measured continuously and arterial oxygen saturation, blood, and saliva samples were assessed after 30 seconds, 3 minutes, and 10 minutes of recovery, respectively. Swimming performance was better with fins than without both with normal frequency breathing and apnea (p < 0.001). Apnea induced no change in lactatemia, but a decrease in arterial oxygen saturation in both SAp and FAp (p < 0.001) was noted and a decrease in HR and swimming performance in SAp (p < 0.01). During apnea without fins, performance alteration was correlated with bradycardia (r = 0.63) and arterial oxygen desaturation (r = -0.57). Saliva dehydroepiandrosterone was increased compared with basal values whatever the trial (p ≤ 0.05), whereas no change was found in saliva cortisol or testosterone. Further studies are necessary to clarify the fin effect on HR and performance during apnea swimming.
Assuntos
Apneia/fisiopatologia , Desempenho Atlético/fisiologia , Lactatos/sangue , Consumo de Oxigênio/fisiologia , Natação/fisiologia , Doença Aguda , Análise de Variância , Atletas , Desidroepiandrosterona/análise , Ensaio de Imunoadsorção Enzimática , Frequência Cardíaca/fisiologia , Humanos , Hidrocortisona/metabolismo , Masculino , Oximetria , Educação Física e Treinamento/métodos , Medição de Risco , Testosterona/análise , Fatores de Tempo , Adulto JovemRESUMO
Luteinizing hormone (LH) is physiologically produced by the anterior pituitary gland. Male athletes may use pharmaceutical LH for doping since it increases the production of testosterone by testes. This hormone is thus on the World Anti-Doping Agency (WADA) list of substances prohibited for males. Anti-doping laboratories perform the assay of this hormone in urine and report abnormally elevated results. We observed a highly significant prevalence of abnormal results in samples taken after a boxing match. Comparison of the descriptive statistics for 426 LH values observed in boxing and other sports showed significant differences. An experimental study comparing urinary LH levels in 17 boxers before and after a match demonstrated a clear increase after the match. The same observation was made for urinary follicle stimulating hormone (FSH) in all of the eight boxers tested for this other pituitary gonadotropin. These observations have consequences for anti-doping controls, as the reference range for urinary LH levels must take into account the specificities of boxers. They also suggest consequences for the health of boxers. Although to our knowledge such observations have never been described, other pituitary disorders have been reported. Our results deserve further investigation from a medical point of view.
Assuntos
Boxe , Hormônio Luteinizante/urina , Dopagem Esportivo , Hormônio Foliculoestimulante/urina , Humanos , Masculino , Testosterona/urinaAssuntos
Adipocinas/sangue , Composição Corporal/efeitos dos fármacos , Glucocorticoides/farmacologia , Aptidão Física/fisiologia , Prednisona/farmacologia , Adiponectina/sangue , Administração Oral , Composição Corporal/fisiologia , Anticoncepcionais Orais/administração & dosagem , Anticoncepcionais Orais/farmacologia , Método Duplo-Cego , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Feminino , Glucocorticoides/administração & dosagem , Humanos , Leptina/sangue , Prednisona/administração & dosagem , Fatores de Tempo , Adulto JovemRESUMO
The purpose of this study was to assess changes in the steroid hormone levels of elite athletes during an international powerlifting competition. Baseline cortisol, DHEA and testosterone were determined in saliva samples in 19 (8 men, 11 women) junior and sub-junior athletes on the day before competition, and then on the competition day during the official weighing and in the hour after competition. Performance was determined by total output and the Wilks formula. No change in saliva steroid concentrations was observed between samples collected on the day before competition and the weighing samples. There was no gender effect on cortisol concentrations but saliva testosterone levels were always significantly higher in men than in women (p<0.01), as was end-competition DHEA (p<0.05). Cortisol and DHEA were significantly increased in male and female athletes after the competition (respectively, p<0.01 and p<0.05), whereas end-competition testosterone concentrations were only significantly increased in men (p<0.01). Significant relationships were demonstrated between performance and end-competition cortisol levels in women and end-competition testosterone levels in men. These data indicate that workouts during an international powerlifting competition produce a significant increase in adrenal steroid hormones in both genders, with an increase in male gonadal steroid hormone. Further studies are necessary to examine the changes in oestradiol and progesterone in women and their potential impact on performance during international powerlifting competition.
Assuntos
Atletas , Hormônios/metabolismo , Internacionalidade , Esteroides/metabolismo , Levantamento de Peso , Exercício Físico , Feminino , Humanos , Masculino , Descanso , Saliva/metabolismo , Fatores de Tempo , Adulto JovemRESUMO
Stimulation of erythropoiesis is one of the most efficient ways of doping. This type of doping is advantageous for aerobic physical exercise and of particular interest to endurance athletes. Erythropoiesis, which takes place in bone marrow, is under the control of EPO, a hormone secreted primarily by the kidneys when the arterial oxygen tension decreases. In certain pathological disorders, such as chronic renal failure, the production of EPO is insufficient and results in anemia. The pharmaceutical industry has, thus, been very interested in developing drugs that stimulate erythropoiesis. With this aim, various strategies have been, and continue to be, envisaged, giving rise to an expanding range of drugs that are good candidates for doping. Anti-doping control has had to deal with this situation by developing appropriate methods for their detection. This article presents an overview of both the drugs and the corresponding methods of detection, and thus follows a roughly chronological order.
Assuntos
Dopagem Esportivo , Eritropoetina/sangue , Hematínicos/sangue , Substâncias para Melhoria do Desempenho/sangue , Proteínas Recombinantes/sangue , Detecção do Abuso de Substâncias/métodos , Anemia/sangue , Animais , Células CHO , Cromatografia Líquida de Alta Pressão/métodos , Cricetinae , Dopagem Esportivo/história , Dopagem Esportivo/métodos , Dopagem Esportivo/tendências , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Eritropoese/efeitos dos fármacos , Eritropoese/fisiologia , Eritropoetina/fisiologia , História do Século XX , História do Século XXI , Humanos , Focalização Isoelétrica/métodos , Falência Renal Crônica/sangue , Masculino , Espectrometria de Massas/métodos , Detecção do Abuso de Substâncias/tendênciasRESUMO
We report here observations clearly demonstrating that gloves can have a deleterious impact on an IEF experiment. These observations were made during the practice of analyses for anti-doping control of erythropoietin. We show that the wearing of nitrile gloves to apply the catholyte strip onto the IEF gel may be responsible for dramatic distortions in the pattern of this hormone. These observations point out that gloves must not be considered only as protective items but also as possible factors in an analytic process.
Assuntos
Luvas Protetoras , Focalização Isoelétrica/normas , Nitrilas/química , Artefatos , Análise de Falha de Equipamento , Eritropoetina/química , Eritropoetina/isolamento & purificação , Humanos , Reprodutibilidade dos TestesRESUMO
Tetracosactide (Synacthen), a synthetic analogue of adrenocorticotropic hormone (ACTH), can be used as a doping agent to increase the secretion of glucocorticoids by adrenal glands. The only published method for anti-doping control of this drug in plasma relies on purification by immunoaffinity chromatography and LC/MS/MS analysis. Its limit of detection is 300 pg/mL, which corresponds to the peak value observed 12 h after 1 mg Synacthen IM administration. We report here a more sensitive method based on preparation of plasma by cation exchange chromatography and solid-phase extraction and analysis by LC/MS/MS with positive-mode electrospray ionization using 7-38 ACTH as internal standard. Identification of Synacthen was performed using two product ions, m/z 671.5 and m/z 223.0, from the parent [M + 5H](5+) ion, m/z 587.4. The recovery was estimated at 70%. A linear calibration curve was obtained from 25 to 600 pg/mL (R² > 0.99). The lower limit of detection was 8 pg/mL (S/N > 3). The lower limit of quantification was 15 pg/mL (S/N > 10; CV% < 20%). The performance of the method was illustrated by an 8-h kinetic analysis of plasma samples from nine subjects submitted to IM injections of either Synacthen® (five subjects) or Synacthen® Depot, the slow-release form of the drug (four subjects). Concentrations of Synacthen between 16 and 310 pg/mL were observed. A sensitive method for quantitation of Synacthen in plasma is proposed for anti-doping control analyses.
Assuntos
Cosintropina/sangue , Cromatografia por Troca Iônica , Cromatografia Líquida , Humanos , Espectrometria de Massas , Sensibilidade e Especificidade , Extração em Fase SólidaRESUMO
OBJECTIVES: Recombinant erythropoietin has a strong impact on aerobic power and is therefore one of the most potent doping agents in endurance sports. The anti-doping control of this synthetic hormone relies on the detection, in the urine, of its isoelectric pattern, which differs from that of the corresponding natural hormone, the latter being typically more acidic than the former. However, a small number of natural urinary patterns, referred to as "atypical patterns," are less acidic than the dominant form. Based on anecdotal evidence, the occurrence of such patterns seems to be related to particular strenuous exercises. This study aimed to demonstrate this relation using a strenuous exercise protocol. DESIGN: Seven athletes took part in a training protocol including a series of supramaximal short-duration exercises. Urine and blood samples were collected throughout the protocols. SETTINGS: World Cycling Center, Aigle, Switzerland, and research laboratories. PARTICIPANTS: Seven top-level athletes (cyclists) were involved in this study. MAIN OUTCOME MEASURES: Erythropoietin (EPO) isoelectric patterns were obtained by submitting blood and urine samples to isoelectric focusing. Additional protein dosages were performed. RESULTS: Supramaximal short-duration exercises induced the transformation of typical urinary natural EPO patterns into atypical ones. None of the obtained atypical patterns fulfilled the 3 criteria mandatory for reporting an adverse analytical finding. Serum EPO patterns were not affected by the exercises that caused the transformation of urinary patterns. CONCLUSION: An exercise-induced transient renal dysfunction is proposed as a hypothetic explanation for these observations that rely on parallel investigations of proteinuria in the same samples.
Assuntos
Eritropoetina/sangue , Eritropoetina/urina , Exercício Físico/fisiologia , Detecção do Abuso de Substâncias/métodos , Adolescente , Adulto , Dopagem Esportivo , Relação Dose-Resposta a Droga , Feminino , Humanos , Focalização Isoelétrica , Masculino , Proteínas Recombinantes , Suíça , Adulto JovemRESUMO
Ex vivo gene therapy is an interesting alternative to orthotopic liver transplantation (OLT) for treating metabolic liver diseases. In this study, we investigated its efficacy and biosafety in nonhuman primates. Hepatocytes isolated from liver lobectomy were transduced in suspension with a bicistronic liver-specific lentiviral vector and immediately autotransplanted (SLIT) into three cynomolgus monkeys. The vector encoded cynomolgus erythropoietin (EPO) and the conditional suicide gene herpes simplex virus-thymidine kinase (HSV-TK). Survival of transduced hepatocytes and vector dissemination were evaluated by detecting transgene expression and vector DNA. SLIT was safely performed within a day in all three subjects. Serum EPO and hematocrit rapidly increased post-SLIT and their values returned to baseline within about 1 month. Isoforms of EPO detected in monkeys' sera differed from the physiological renal EPO. In liver biopsies at months 8 and 15, we detected EPO protein, vector mRNA and DNA, demonstrating long-term survival and functionality of transplanted lentivirally transduced hepatocytes. Valganciclovir administration resulted in complete ablation of the transduced hepatocytes. We demonstrated the feasibility and biosafety of SLIT, and the long term (>1 year) functionality of lentivirally transduced hepatocytes in nonhuman primates. The HSV-TK/valganciclovir suicide strategy can increase the biosafety of liver gene therapy protocols by safely and completely ablating transduced hepatocytes on demand.
Assuntos
Terapia Genética/métodos , Hepatócitos/virologia , Lentivirus/genética , Transdução Genética/métodos , Animais , Antivirais/farmacologia , Western Blotting , Linhagem Celular , Células Cultivadas , Eritropoetina/genética , Eritropoetina/fisiologia , Ganciclovir/análogos & derivados , Ganciclovir/farmacologia , Vetores Genéticos/genética , Células HeLa , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Hepatopatias/terapia , Macaca fascicularis , Masculino , Reação em Cadeia da Polimerase , Simplexvirus/genética , Timidina Quinase/genética , Timidina Quinase/fisiologia , Valganciclovir , Proteínas Virais/genética , Proteínas Virais/fisiologiaRESUMO
Nonspecific interactions between blotted proteins and unrelated secondary antibodies generate false positives in immunoblotting techniques. Some procedures have been developed to reduce this adsorption but they may work in specific applications and be ineffective in other ones. "Double-blotting" has been developed to overcome this problem. It consists of interpolating a second blotting step between the usual probings of the blot membrane with the primary antibody and the secondary antibodies. This step, by isolating the primary antibody from the interfering proteins, guarantees the specificity of the probing with the secondary antibody. This method has been developed for the study of erythropoietin in concentrated urine since a strong nonspecific binding of biotinylated secondary antibodies to some urinary proteins is observed using classical immunoblotting protocols. However, its concept makes it usable in other applications that come up against this kind of problem. This method is expected to be especially useful for investigating proteins that are present in minute amounts in complex biological media.