RESUMEN
The successful use of homogenized horse liver for the generation of phase I in vitro metabolites has been previously reported by the authors' laboratory. Prior to the use of homogenized liver, the authors' laboratory had been using mainly horse liver microsomes for carrying out equine in vitro metabolism studies. Homogenized horse liver has shown significant advantages over liver microsomes for in vitro metabolism studies as the procedures are much quicker and have higher capability for generating more in vitro metabolites. In this study, the use of homogenized liver has been extended to the generation of phase II in vitro metabolites (glucuronide and/or sulfate conjugates) using 17ß-estradiol, morphine, and boldenone undecylenate as model substrates. It was observed that phase II metabolites could also be generated even without the addition of cofactors. To the authors' knowledge, this is the first report of the successful use of homogenized horse liver for the generation of phase II metabolites. It also demonstrates the ease with which both phase I and phase II metabolites can now be generated in vitro simply by using homogenized liver without the need for ultracentrifuges or tedious preparation steps.
Asunto(s)
Hígado/química , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Estradiol/metabolismo , Glucurónidos/metabolismo , Caballos , Indicadores y Reactivos , Hígado/metabolismo , Espectrometría de Masas , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Morfina/metabolismo , Sulfatos/metabolismo , Testosterona/análogos & derivados , Testosterona/metabolismoRESUMEN
1-Testosterone (17ß-hydroxy-5α-androst-1-en-3-one), a synthetic anabolic steroid, has been described as one of the most effective muscle-building supplements currently on the market. It has an anabolic potency of 200 as compared to 26 for testosterone. Apart from its abuse in human sports, it can also be a doping agent in racehorses. Metabolic studies on 1-testosterone have only been reported for human in the early seventies, whereas little is known about its metabolic fate in horses. This paper describes the studies of in vitro and in vivo metabolism of 1-testosterone in horses, with the aim of identifying the most appropriate target metabolites to be monitored for controlling the misuse or abuse of 1-testosterone in racehorses. Six in vitro metabolites, namely 5α-androst-1-ene-3α,17ß-diol (T1a), 5α-androstane-3ß,17ß-diol (T2), epiandrosterone (T3), 16,17-dihydroxy-5α-androst-1-ene-3-one (T4 & T5), and 5α-androst-1-ene-3,17-dione (T6), were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 800 mg of 1-testosterone by stomach tubing. The results revealed that the parent drug and eight metabolites were detected in urine. Besides the four in vitro metabolites (T1a, T2, T3, and T5), four other urinary metabolites, namely 5α-androst-1-ene-3ß,17α-diol (T1b), 5α-androst-1-ene-3ß,17ß-diol (T1c), 5α-androstane-3α,17α-diol (T7) and 5α-androstane-3ß,17α-diol (T8) were identified. This study shows that the detection of 1-testosterone administration is best achieved by monitoring the parent drug, which could be detected for up to 30 h post-administration.
Asunto(s)
Anabolizantes/metabolismo , Caballos/metabolismo , Testosterona/análogos & derivados , Anabolizantes/orina , Animales , Cromatografía de Gases y Espectrometría de Masas , Caballos/orina , Microsomas Hepáticos/metabolismo , Testosterona/metabolismoRESUMEN
Gas chromatography-mass spectrometry (GC-MS) analysis after heptafluorobutyric anhydride (HFBA) derivatization was one of the published methods used for the quantification of ephedrine (EP) and pseudoephedrine (PE) in urine. This method allows the clear separation of the derivatized diastereoisomers on a methyl-silicone-based column. Recently the authors came across a human urine sample with apparently high levels (µg/ml) of EP and PE upon initial screening. However, duplicate analyses of this sample using the HFBA-GC-MS method revealed an unusual discrepancy in the estimated levels of EP and PE, with the area response ratios of EP/PE at around 29% on one occasion and around 57% on another. The same sample was re-analyzed for EP and PE using other techniques, including GC-MS after trimethylsilylation and ultra-high-performance liquid chromatography-tandem mass spectrometry. Surprisingly, the concentration of EP in the sample was determined to be at least two orders of magnitude less than what was observed with the HFBA-GC-MS method. A thorough investigation was then conducted, and the results showed that both substances could interconvert during HFBA derivatization. Similar diastereoisomeric conversion was also observed using other fluorinated acylating agents (e.g. pentafluoropropionic anhydride and trifluoroacetic anhydride). The extent of interconversion was correlated with the degree of fluorination of the acylating agents, with HFBA giving the highest conversion. This conversion has never been reported before. A mechanism for the interconversion was proposed. These findings indicated that fluorinated acylating agents should not be used for the unequivocal identification or quantification of EP and PE as the results obtained can be erroneous.
Asunto(s)
Efedrina/química , Fluorocarburos/química , Sustancias para Mejorar el Rendimiento/química , Seudoefedrina/química , Anhídridos Acéticos/química , Acilación , Artefactos , Cromatografía Líquida de Alta Presión , Efedrina/orina , Fluoroacetatos/química , Cromatografía de Gases y Espectrometría de Masas , Humanos , Isomerismo , Modelos Químicos , Sustancias para Mejorar el Rendimiento/orina , Seudoefedrina/orina , Reproducibilidad de los Resultados , Detección de Abuso de Sustancias/métodos , Espectrometría de Masas en TándemRESUMEN
Liquid chromatography/mass spectrometry (LC/MS) has been successfully applied to the detection of anabolic steroids in biological samples. However, the sensitive detection of saturated hydroxysteroids, such as androstanediols, by electrospray ionisation (ESI) is difficult because of their poor ability to ionise. In view of this, chemical derivatisation has been used to enhance the detection sensitivity of hydroxysteroids by LC/MS. This paper describes the development of a sensitive ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) method for the screening of anabolic steroids in horse urine by incorporating a chemical derivatisation step, using picolinic acid as the derivatisation reagent. The method involved solid-phase extraction (SPE) of both free and conjugated anabolic steroids in horse urine using a polymer-based SPE cartridge (Abs Elut Nexus). The conjugated steroids in the eluate were hydrolysed by methanolysis and the resulting extract was further cleaned up by liquid-liquid extraction. The resulting free steroids in the extract were derivatised with picolinic acid to form the corresponding picolinoyl esters and analysed by UHPLC/MS/MS in the positive ESI mode with selected-reaction-monitoring. Separation of the targeted steroids was performed on a C18 UHPLC column. The instrument turnaround time was 10.5 min inclusive of post-run equilibration. A total of thirty-three anabolic steroids (including 17ß-estradiol, 5(10)-estrene-3ß,17α-diol, 5α-estrane-3ß,17α-diol, 17α-ethyl-5α-estran-3α,17ß-diol, 17α-methyl-5α-androstan-3,17ß-diols, androstanediols, nandrolone and testosterone) spiked in negative horse urine at the QC levels (ranging from 0.75 to 30 ng/mL) could be consistently detected. The intra-day and inter-day precisions (% RSD) for the peak area ratios were around 7-51% and around 1-72%, respectively. The intra-day and inter-day precisions (% RSD) for the relative retention times were both less than 1% for all analytes, except the inter-day precision for boldione at 1.2%. The extraction recoveries for all targets were not less than 48%. With exceptional separation achieved by the UHPLC system, matrix interferences were minimal at the expected retention times of the selected transitions. As detection was performed with an UHPLC system coupled to a fast-scanning triple quadrupole mass spectrometer, the method could easily be expanded to accommodate additional steroid targets. This method has been validated for recovery and precision, and could be used regularly for doping control testing of anabolic steroids in horse urine samples.
Asunto(s)
Anabolizantes/orina , Cromatografía Líquida de Alta Presión/métodos , Hidroxiesteroides/orina , Espectrometría de Masas en Tándem/métodos , Anabolizantes/química , Animales , Doping en los Deportes , Caballos , Hidroxiesteroides/química , Masculino , Metanol , Ácidos Picolínicos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Extracción en Fase SólidaRESUMEN
The authors' laboratory at one time employed four liquid chromatography/mass spectrometric (LC/MS) methods for the detection of a large variety of drugs in equine urine. Drug classes covered by these methods included anti-diabetics, anti-ulcers, cyclooxygenase-2 (COX-2) inhibitors, sedatives, corticosteroids, anabolic steroids, sulfur diuretics, xanthines, etc. With the objective to reduce labour and instrumental workload, a new ultra performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method has been developed, which encompasses all target analytes detected by the original four LC/MS methods. The new method has better detection limits than the superseded methods. In addition, it covers new target analytes that could not be adequately detected by the four LC/MS methods. The new method involves solid-phase extraction (SPE) of two aliquots of equine urine using two Abs Elut Nexus cartridges. One aliquot of the urine sample is treated with ß-glucuronidase before subjecting to SPE. A second aliquot of the same urine sample is processed directly using another SPE cartridge, so that drugs that are prone to decomposition during enzyme hydrolysis can be preserved. The combined eluate is analysed by UPLC/MS/MS using alternating positive and negative electrospray ionisation in the selected-reaction-monitoring mode. Exceptional chromatographic separation is achieved using an UPLC system equipped with a UPLC(®) BEH C18 column (10 cm L×2.1 mm ID with 1.7 µm particles). With this newly developed UPLC/MS/MS method, the simultaneous detection of 140 drugs at ppb to sub-ppb levels in equine urine can be achieved in less than 13 min inclusive of post-run equilibration. Matrix interference for the selected transitions at the expected retention times is minimised by the excellent UPLC chromatographic separation. The method has been validated for recovery and precision, and is being used regularly in the authors' laboratory as an important component of the array of screening methods for doping control analyses of equine urine samples.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Glucuronidasa/metabolismo , Caballos/orina , Péptido Hidrolasas/metabolismo , Preparaciones Farmacéuticas/orina , Espectrometría de Masas en Tándem/métodos , Urinálisis/métodos , Animales , Bovinos , Cromatografía Líquida de Alta Presión/economía , Doping en los Deportes , Hidrólisis , Rótula/enzimología , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/aislamiento & purificación , Preparaciones Farmacéuticas/metabolismo , Extracción en Fase Sólida , Espectrometría de Masas en Tándem/economía , Factores de Tiempo , Urinálisis/economíaRESUMEN
The study of the metabolism of drugs, in particular steroids, by both in vitro and in vivo methods has been carried out in the authors' laboratory for many years. For in vitro metabolic studies, the microsomal fraction isolated from horse liver is often used. However, the process of isolating liver microsomes is cumbersome and tedious. In addition, centrifugation at high speeds (over 100 000 g) may lead to loss of enzymes involved in phase I metabolism, which may account for the difference often observed between in vivo and in vitro results. We have therefore investigated the feasibility of using homogenized horse liver instead of liver microsomes with the aim of saving preparation time and improving the correlation between in vitro and in vivo results. Indeed, the preparation of the homogenized horse liver was very simple, needing only to homogenize the required amount of liver. Even though no further purification steps were performed before the homogenized liver was used, the cleanliness of the extracts obtained, based on gas chromatography-mass spectrometry (GC-MS) analysis, was similar to that for liver microsomes. Herein, the results of the in vitro experiments carried out using homogenized horse liver for five anabolic steroids-turinabol, methenolone acetate, androst-4-ene-3,6,17-trione, testosterone, and epitestosterone-are discussed. In addition to the previously reported in vitro metabolites, some additional known in vivo metabolites in the equine could also be detected. As far as we know, this is the first report of the successful use of homogenized liver in the horse for carrying out in vitro metabolism experiments. Copyright © 2011 John Wiley & Sons, Ltd.
Asunto(s)
Extractos Hepáticos/metabolismo , Microsomas Hepáticos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Andrógenos/análisis , Andrógenos/metabolismo , Androstenos/análisis , Androstenos/metabolismo , Animales , Biotransformación , Epitestosterona/análisis , Epitestosterona/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Caballos , Técnicas In Vitro , Hígado/metabolismo , Metenolona/análogos & derivados , Metenolona/análisis , Metenolona/metabolismo , Estructura Molecular , Preparaciones Farmacéuticas/análisis , Testosterona/análogos & derivados , Testosterona/análisis , Testosterona/metabolismoRESUMEN
Currently there are two common radioimmunoassay-based methods for the detection of equine cryptorchidism; one measures testosterone concentrations in peripheral blood samples taken before and after an intravenous injection of human chorionic gonadotrophin (hCG) and the other measures plasma estrone sulfate. However, each of these invasive methods has its own shortfalls and neither gives unequivocal results. In this article a highly reliable gas chromatography/mass spectrometry (GC/MS) method is described based on the analysis of urine samples for the identification of cryptorchidism in horses, some as young as 2 years old.
Asunto(s)
Criptorquidismo/veterinaria , Cromatografía de Gases y Espectrometría de Masas/veterinaria , Enfermedades de los Caballos/orina , Esteroides/orina , Urinálisis/veterinaria , Animales , Criptorquidismo/diagnóstico , Criptorquidismo/orina , Enfermedades de los Caballos/diagnóstico , Caballos , MasculinoRESUMEN
This paper describes the application of gas chromatography-mass spectrometry (GC-MS) for in vitro and in vivo studies of 6-OXO in horses, with a special aim to identify the most appropriate target metabolite to be monitored for controlling the administration of 6-OXO in racehorses. In vitro studies of 6-OXO were performed using horse liver microsomes. The major biotransformation observed was reduction of one keto group at the C3 or C6 positions. Three in vitro metabolites, namely 6alpha-hydroxyandrost-4-ene-3,17-dione (M1), 3alpha-hydroxyandrost-4-ene-6,17-dione (M2a) and 3beta-hydroxyandrost-4-ene-6,17-dione (M2b) were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 500 mg of androst-4-ene-3,6,17-trione (5 capsules of 6-OXO((R))) by stomach tubing. The results revealed that 6-OXO was extensively metabolized. The three in vitro metabolites (M1, M2a and M2b) identified earlier were all detected in post-administration urine samples. In addition, seven other urinary metabolites, derived from a further reduction of either one of the remaining keto groups or one of the remaining keto groups and the olefin group, were identified. These metabolites included 6alpha,17beta-dihydroxyandrost-4-en-3-one (M3a), 6,17-dihydroxyandrost-4-en-3-one (M3b and M3c), 3beta,6beta-dihydroxyandrost-4-en-17-one (M4a), 3,6-dihydroxyandrost-4-en-17-one (M4b), 3,6-dihydroxyandrostan-17-one (M5) and 3,17-dihydroxyandrostan-6-one (M6). The longest detection time observed in urine was up to 46 h for the M6 metabolite. For blood samples, the peak 6-OXO plasma concentration was observed 1 h post administration. Plasma 6-OXO decreased rapidly and was not detectable 12 h post administration.
Asunto(s)
Anabolizantes/análisis , Anabolizantes/orina , Androstenos/análisis , Androstenos/orina , Cromatografía de Gases y Espectrometría de Masas/métodos , Caballos/orina , Anabolizantes/metabolismo , Androstenos/metabolismo , Animales , Doping en los Deportes , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismoRESUMEN
In September 2005, our laboratory detected the presence of 4-androstene-3,17-dione and androsterone in a standard steroid screen of a post-race gelding urine sample received from an overseas authority. All other urine samples from the same batch tested negative. Subsequent gas chromatography/mass spectrometry (GC/MS) confirmatory analyses, however, repeatedly failed to detect any amount of 4-androstene-3,17-dione and androsterone in the suspicious sample. On the other hand, identical results were obtained when the initial GC/MS screening method was repeated on the suspicious sample as well as on the other samples of the same batch, showing the presence of 4-androstene-3,17-dione and androsterone only in the suspicious sample. These unusual and contradictory findings between the screening and confirmatory procedures were investigated, leading to the unequivocal conclusion that the 4-androstene-3,17-dione and androsterone observed during screening were artefacts from the internal standards, [16,16,17-d3]-testosterone and [16,16,17-d3]-5alpha-androstane-3alpha,17beta-diol. The two deuterated internal standards were thought to have undergone first an enzymatic oxidation of the 17beta-hydroxyl group to a 17-keto function by the enzyme 17beta-hydroxysteroid dehydrogenase; complete deuterium-hydrogen exchange at C16 during the methanolysis deconjugation step would then produce the two artefacts. The findings from this study highlight the potential problem of using internal standards in qualitative confirmatory analyses, which may lead to undesirable false positive results.
Asunto(s)
Anabolizantes/orina , Androstenodiona/orina , Androsterona/orina , Doping en los Deportes , Toxicología Forense , Detección de Abuso de Sustancias/métodos , Animales , Artefactos , Cromatografía de Gases y Espectrometría de Masas , Caballos , Técnicas In Vitro , Masculino , Microsomas Hepáticos/metabolismo , Reproducibilidad de los Resultados , UrinálisisRESUMEN
A multi-target high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of low ppt to low ppb levels of anabolic steroids, corticosteroids, anti-diabetics, and non-steroidal anti-inflammatory drugs (NSAIDs) in equine plasma was developed for the purpose of doping control. Plasma samples were first deproteinated by addition of trichloroacetic acid. Drugs were then extracted by solid-phase extraction (SPE) using Bond Elut Certify cartridges, and the extracts were analysed by a triple-quadrupole/linear ion trap LC-MS-MS instrument in positive electrospray ionization (+ESI) mode with selected reaction monitoring (SRM) scan function. Chromatographic separation of the targeted drugs was achieved using a reverse phase 3.3 cm L x 2.1 mm ID, 3 microm particle size LC column with gradient elution. Plasma samples fortified with 66 targeted drugs including betamethasone, boldione, capsaicin, flunisolide, gestrinone, gliclazide, 17alpha-hydroxyprogesterone hexanoate, isoflupredone and triamcinolone acetonide, etc. at low ppt to low ppb levels could be consistently detected. No significant matrix interference was observed at the retention time of the targeted ion transitions when blank plasma samples were analysed. The method has been validated for its extraction recoveries, precision and sensitivity, and is used regularly in the authors' laboratory to screen for the presence of these drugs in plasma samples from racehorses.
Asunto(s)
Anabolizantes/sangre , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Caballos , Hipoglucemiantes/sangre , Reproducibilidad de los Resultados , Detección de Abuso de Sustancias/métodosRESUMEN
Quantitative determination, particularly for threshold substances in biological samples, is much more demanding than qualitative identification. A proper assessment of any quantitative determination is the measurement uncertainty (MU) associated with the determined value. The International Standard ISO/IEC 17025, "General requirements for the competence of testing and calibration laboratories", has more prescriptive requirements on the MU than its superseded document, ISO/IEC Guide 25. Under the 2005 or 1999 versions of the new standard, an estimation of the MU is mandatory for all quantitative determinations. To comply with the new requirement, a protocol was established in the authors' laboratory in 2001. The protocol has since evolved based on our practical experience, and a refined version was adopted in 2004. This paper describes our approach in establishing the MU, as well as some other important considerations, for the quantification of threshold substances in biological samples as applied in the area of doping control for horses. The testing of threshold substances can be viewed as a compliance test (or testing to a specified limit). As such, it should only be necessary to establish the MU at the threshold level. The steps in a "Bottom-Up" approach adopted by us are similar to those described in the EURACHEM/CITAC guide, "Quantifying Uncertainty in Analytical Measurement". They involve first specifying the measurand, including the relationship between the measurand and the input quantities upon which it depends. This is followed by identifying all applicable uncertainty contributions using a "cause and effect" diagram. The magnitude of each uncertainty component is then calculated and converted to a standard uncertainty. A recovery study is also conducted to determine if the method bias is significant and whether a recovery (or correction) factor needs to be applied. All standard uncertainties with values greater than 30% of the largest one are then used to derive the combined standard uncertainty. Finally, an expanded uncertainty is calculated at 99% one-tailed confidence level by multiplying the standard uncertainty with an appropriate coverage factor (k). A sample is considered positive if the determined concentration of the threshold substance exceeds its threshold by the expanded uncertainty. In addition, other important considerations, which can have a significant impact on quantitative analyses, will be presented.
Asunto(s)
Caballos/orina , Detección de Abuso de Sustancias/métodos , Detección de Abuso de Sustancias/veterinaria , Algoritmos , Animales , Calibración , Técnicas de Laboratorio Clínico/normas , Modelos Teóricos , Control de Calidad , Estándares de Referencia , Detección de Abuso de Sustancias/normasRESUMEN
This paper describes two high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for the screening of two important classes of drugs in equine sports, namely corticosteroids and basic drugs, at low ppb levels in horse urine. The method utilized a high efficiency reversed-phase LC column (3.3 cm L x 2.1 mm i.d. with 3 microm particles) to provide fast turnaround times. The overall turnaround time for the corticosteroid screen was 5 min and that for the basic drug screen was 8 min, inclusive of post-run and equilibration times. Method specificity was assessed by analysing a total of 35 negative post-race horse urine samples. No interference from the matrices at the expected retention times of the targeted masses was observed. Inter-day precision for the screening of 19 corticosteroids and 48 basic drugs were evaluated by replicate analyses (n = 10) of a spiked sample on 4 consecutive days. The results demonstrated that both methods have acceptable precision to be used on a routine basis. The performance of these two methods on real samples was demonstrated by their applications to drug administration and positive post-race urine samples.
Asunto(s)
Corticoesteroides/orina , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Preparaciones Farmacéuticas/orina , Animales , Doping en los Deportes , Caballos , Masculino , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Accurate measurements of airway and lesion dimensions are important to the developmental progress of paediatric bronchoscopy. The malacia disorders are an important cause of respiratory morbidity in children, but no methods are currently available to measure these lesions or the airway lumen accurately. A new measurement technique is described here. METHODS: The magnification power of a paediatric videobronchoscope was defined and a simple and user friendly computer based program (Image J) was used to develop an objective technique (colour histogram mode technique, CHMT) for measurement of the airway lumen. RESULTS: In vivo intra-observer and inter-observer repeatability coefficients for repeated area measurements from 28 images using the Bland-Altman method were 0.9 mm2 and 1.6 mm2, respectively. The average intraclass correlation coefficient for repeated measurements of area was 0.93. In vitro validation measurements using a 2 mm diameter tube resolved radii measurements to within 0.1 mm (coefficient of variability 8%). An "acceptable result" was defined in 92% of 734 images completed with the CHMT alone and 8% with its modification. The success rate for two of three images being within 10% of each other's area was 100%. Measurements of cricoid cross sectional areas from 116 patients compared with expected airway areas for age derived from endotracheal tube sizes were comparable. CONCLUSIONS: The CHMT method of identifying and measuring airway dimensions is objective, accurate, and versatile and, as such, is important to the future development of flexible videobronchoscopy.
Asunto(s)
Bronquios/anatomía & histología , Broncoscopía/métodos , Broncoscopios/normas , Broncoscopía/normas , Calibración , Niño , Color , Humanos , Microscopía por Video/métodos , Microscopía por Video/normas , Variaciones Dependientes del Observador , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Fatty acid-binding protein (FABP) holds promise for early detection of tissue injury. This small protein (15kD) appears earlier in the blood than large proteins after cell damage. Combined its characteristics of high concentration tissue contents and low normal plasma values provide the possibility of a rapid rise above the respective reference values, and thus an early indication of the appearance of tissue injury. A general review was presented on the current status of different types of FABP for the detection of tissue injury in patients with myocardial injury, brain injury and also in athletes or horses with skeletal muscle injury. To take full advantage of the characteristics of the early marker FABP, rapid analysis is a crucial parameter. In this review, an overview of the development of immunoassay for the quantification of FABP in buffer, plasma or whole blood was outlined. The characteristics of different FABP immunosensors and immunotests were described. The feasibility of these immunoassays to be used in routine clinical practice and in emergency case was also discussed. Nowadays, the improved automated immunoassays (e.g. a microparticle-enhanced turbidimetric immunoassay), less time-consuming bedside immunosensors and immunotests (e.g. a one-step FABP lateral flow immunotest), are the main advance technology in point-of-care testing. With these point-of-care tests, the application of FABP as an early tissue injury marker has a great potential for many clinical purposes.
Asunto(s)
Técnicas Biosensibles/instrumentación , Análisis Químico de la Sangre/instrumentación , Proteínas Portadoras/sangre , Enfermedades del Tejido Conjuntivo/sangre , Enfermedades del Tejido Conjuntivo/diagnóstico , Inmunoensayo/instrumentación , Monitoreo Fisiológico/instrumentación , Animales , Biomarcadores/sangre , Técnicas Biosensibles/métodos , Análisis Químico de la Sangre/métodos , Diseño de Equipo , Proteínas de Unión a Ácidos Grasos , Humanos , Inmunoensayo/métodos , Monitoreo Fisiológico/métodos , Infarto del Miocardio/sangre , Infarto del Miocardio/diagnósticoRESUMEN
BACKGROUND: Flexible video bronchoscopes, in particular the Olympus BF Type 3C160, are commonly used in pediatric respiratory medicine. There is no data on the magnification and distortion effects of these bronchoscopes yet important clinical decisions are made from the images. The aim of this study was to systematically describe the magnification and distortion of flexible bronchoscope images taken at various distances from the object. METHODS: Using images of known objects and processing these by digital video and computer programs both magnification and distortion scales were derived. RESULTS: Magnification changes as a linear function between 100 mm (x1) and 10 mm (x9.55) and then as an exponential function between 10 mm and 3 mm (x40) from the object. Magnification depends on the axis of orientation of the object to the optic axis or geometrical axis of the bronchoscope. Magnification also varies across the field of view with the central magnification being 39% greater than at the periphery of the field of view at 15 mm from the object. However, in the paediatric situation the diameter of the orifices is usually less than 10 mm and thus this limits the exposure to these peripheral limits of magnification reduction. Intraclass correlations for measurements and repeatability studies between instruments are very high, r = 0.96. Distortion occurs as both barrel and geometric types but both types are heterogeneous across the field of view. Distortion of geometric type ranges up to 30% at 3 mm from the object but may be as low as 5% depending on the position of the object in relation to the optic axis. CONCLUSION: We conclude that the optimal working distance range is between 40 and 10 mm from the object. However the clinician should be cognisant of both variations in magnification and distortion in clinical judgements.
Asunto(s)
Artefactos , Broncoscopios , Interpretación de Imagen Asistida por Computador/métodos , Microscopía por Video/instrumentación , Pediatría/instrumentación , Análisis de Falla de Equipo , Aumento de la Imagen/instrumentación , Aumento de la Imagen/métodos , Microscopía por Video/métodos , Fantasmas de Imagen , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Boldenone (1,2-dehydrotestosterone) is a common veterinary anabolic agent. Its structure is very similar to testosterone. Testosterone is endogenous in the horse, whereas there has been no report concerning the detection of endogenous boldenone. This paper reports the direct observation of sulphate conjugate of boldenone in equine urine from entires. The detection procedures involved solid-phase extraction, immunoaffinity column (IAC) purification, and then LC-MS-MS analysis on a Q-ToF instrument. The identification of boldenone sulphate has provided direct evidence for the endogenous nature of boldenone in entire male horses. Quantification data for the normal level of boldenone in Hong Kong racehorses will also be discussed.
Asunto(s)
Cromatografía Liquida/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Espectrometría de Masas/métodos , Testosterona/análogos & derivados , Testosterona/orina , Animales , Caballos , MasculinoRESUMEN
Laryngomalacia, bronchomalacia, and tracheomalacia are commonly seen in pediatric respiratory medicine, yet their patterns and associations with other conditions are not well-understood. We prospectively video-recorded bronchoscopic data and clinical information from referred patients over a 10-year period and defined aspects of interrelationships and associations. Two hundred and ninety-nine cases of malacia disorders (34%) were observed in 885 bronchoscopic procedures. Cough, wheeze, stridor, and radiological changes were the most common symptoms and signs. The lesions were most often found in males (2:1) and on the left side (1.6:1). Concomitant malacia lesions ranged from 24% for laryngotracheobronchomalacia to 47% for tracheobronchomalacia. The lesions were found in association with other disorders such as congenital heart disorders (13.7%), tracheo-esophageal fistula (9.6%), and various syndromes (8%). Even though the understanding of these disorders is in its infancy, pediatricians should maintain a level of awareness for malacia lesions and consider the possibility of multiple lesions being present, even when one symptom predominates or occurs alone.
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Enfermedades Respiratorias/epidemiología , Adolescente , Adulto , Enfermedades Bronquiales/diagnóstico , Enfermedades Bronquiales/epidemiología , Broncoscopía , Niño , Preescolar , Comorbilidad , Femenino , Humanos , Incidencia , Lactante , Recién Nacido , Enfermedades de la Laringe/diagnóstico , Enfermedades de la Laringe/epidemiología , Masculino , Enfermedades Respiratorias/diagnóstico , Enfermedades de la Tráquea/diagnóstico , Enfermedades de la Tráquea/epidemiologíaRESUMEN
OBJECTIVE: The aim of this study was to describe and compare the nutritional status of children aged 0-18 years attending the cystic fibrosis (CF) clinic at the Royal Children's Hospital, Brisbane, Australia, as outpatients in 1986 and 1996. METHODOLOGY: The heights, weights and pulmonary function of children attending the CF clinic as outpatients in 1986 (n = 97) and 1996 (n = 227) were retrospectively analysed using a computerized database maintained by the CF clinic. The heights and weights were analysed in terms of z scores for height for age (HAZ), weight for age (WAZ) and weight for height (WHZ). Pulmonary function data is not available for all children. Forced expiratory volume in one second (FEV1), forced vital capacity (FVC) and forced mid expiratory flows (FEF) were expressed as a percentage of predicted and are presented here. RESULTS: The 1986 sample consisted of 41 males (age range 0.18-14.59 years, mean age 6.52 (4.33)) and 56 females (age range 0.15-14.97 years, mean age 7.75 (3.70)). The 1996 sample consisted of 111 males (age range 0.09-17.97 years, mean age 8.80 (5.49)) and 114 females (age range 0.12-17.98 years, mean age 8.49 (5.26)). In 1986, males were shorter than females (P = 0.0096) and females had a lower mean FVC than males (P = 0.0438). In 1996, males were shorter, lighter and more wasted than females (P = 0.0357, P = 0.0034 and P = 0.0273, respectively) and females had a lower mean FEV1 and mean FVC than males (P = 0.0176 and P = 0.0079, respectively). Males in 1996 were lighter and more wasted than males in 1986 (P = 0.0023 and P = 0.0139, respectively) and had a lower mean FEV1, mean FVC and mean FEF (P < 0.0001, P = 0.0012 and P = 0.0069, respectively). Females in 1996 were shorter and lighter than females in 1986 (P = 0.0273 and P = 0.0405, respectively) and had a lower mean FEV1, mean FVC and mean FEF (P < 0.0001, P < 0.0001 and P < 0.0001, respectively). When subjects were classified according to FEV1 (FEV1 > or = 75% or FEV1 < 75%), there were no significant differences in z scores between the 1986 group and 1996 group. Similarly, when the 1986 group were matched for gender and FEV1 with the 1996 group, there were no significant differences in z scores for males or females. CONCLUSIONS: It is suggested that the apparent worsening of nutritional status among the 1996 group of CF patients is in fact due to an effect of increased survival of patients with more severe clinical symptoms. The findings from this study highlight the continuing, and in fact, worsening problem of growth failure in children with CF.
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Fibrosis Quística/complicaciones , Trastornos del Crecimiento/etiología , Estado Nutricional , Adolescente , Atención Ambulatoria/tendencias , Estatura , Peso Corporal , Niño , Preescolar , Fibrosis Quística/fisiopatología , Femenino , Volumen Espiratorio Forzado , Trastornos del Crecimiento/diagnóstico , Humanos , Lactante , Masculino , Flujo Espiratorio Medio Máximo , Queensland , Valores de Referencia , Estudios Retrospectivos , Análisis de Supervivencia , Capacidad VitalRESUMEN
OBJECTIVE: To compare the efficacy of salbutamol delivered by jet nebuliser (JN) with salbutamol via a pressurised metered dose inhaler (PMDI) and a large volume spacer (Volumatic) for management of acute asthma. STUDY POPULATION: A total of 160 children aged from 4 to 12 years presenting to an Emergency Department with acute asthma. METHODS: The study was of multicentre (n=5) randomised, double blind, parallel design. Children weighing less than 25 kg received salbutamol 2.5 mg via the JN or 600 microg (six puffs) from the PMDI. Children over 25 kg received salbutamol 5 mg via the JN or 1200 microg (12 puffs) via the PMDI. Clinical score (range 0-12) and PEF (over 7 years) were recorded at baseline and 15, 30, 45 and 60 mins post administration. RESULTS: The improvement from baseline at 30 min in the clinical score was 1.87 for JN and 1.43 for PMDI (P=0.09) and at 60 min was 2.15 for JN and 1.12 for PMDI (P=0.0001). The improvement in PEF at 30 min was 51 L min(-1) for JN and 27 L min(-1) for PMDI (P=0.0007) and at 60 min was 57 L min(-1) for JN and 31.5 L min(-1) for PMDI (P=0.001). CONCLUSION: Administration of salbutamol via a PMDI and a large volume spacer device provides effective relief in the management of acute asthma in children, but to a lesser extent than a jet nebuliser. This difference may represent a dose response effect.
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Albuterol/administración & dosificación , Asma/tratamiento farmacológico , Broncodilatadores/administración & dosificación , Nebulizadores y Vaporizadores , Enfermedad Aguda , Administración por Inhalación , Aerosoles , Niño , Preescolar , Método Doble Ciego , Femenino , Humanos , MasculinoRESUMEN
Atypical mycobacterial infection in HIV-negative children usually presents with cervical lymphadenopathy. We report on 10 children who are HIV-negative and who presented with pulmonary disease, in whom either culture-proven atypical mycobacterium infection (four), positive avian Mantoux test (five), or lack of response to human tuberculosis treatment (one) had been observed. One case was subsequently diagnosed as chronic granulomatous disease and illustrates that children with atypical mycobacterial pulmonary infection should have their immune status fully investigated. Bronchial obstruction was observed in eight cases, and of these, endobronchial disease was found in six children. The diagnosis of atypical mycobacterial disease is difficult, and a negative avian Mantoux test does not exclude the diagnosis. The availability of clarithromycin and rifabutin has offered new therapeutic options in treating atypical mycobacterial pulmonary infection, but management of these cases can be prolonged and difficult.