RESUMEN
Canine reproduction differs from that of many other domestic animals, and increased knowledge on biochemical changes during canine pregnancy is important for investigations of infertility or subfertility. The total glycosylation pattern, i.e., the glycome, of body fluids reflects cellular status in health and disease. The aim of the present pilot study was to investigate pregnancy-related changes of the serum N-glycome in bitches. A method based on Rapifluor HILIC-UPLC-FLR-MS was optimized and applied for analysis and quantification of N-glycans in canine serum. Serum samples from six pregnant and five non-pregnant bitches, collected at four well-defined time points, were included. The levels of sialylated and galactosylated complex glycans were significantly elevated in serum from pregnant bitches, consistent with previous reports on human pregnancy. The levels of fucosylated and agalactosylated glycans decreased significantly in pregnant dogs. In non-pregnant dogs, the glycosylation pattern did not change during the cycle. Pregnancy is an inflammatory state, but our findings during canine pregnancy are quite the opposite to changes that have previously been described for dogs with a known parasitic infection. Evaluation of the canine glycome may thus be valuable in studies of canine pregnancy, possibly differing inflammatory changes related to pregnancy to those caused by an infection.
Asunto(s)
Polisacáridos , Animales , Perros , Femenino , Glicosilación , Embarazo , Polisacáridos/sangre , Polisacáridos/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Preñez/sangre , Espectrometría de Masas/métodos , Proyectos PilotoRESUMEN
Erythropoiesis stimulating agents (ESAs) are a group of therapeutic glycoproteins used to treat anaemia caused by chronic kidney disease or chemotherapy. A variety of ESA products are available in the European Union, including innovator, biosimilar and second-generation medicines. Glycosylation is a critical quality attribute of ESA products, as it has a crucial influence upon in vivo biological activity. In this study, a combination of chromatography and mass spectrometry analysis has been used to characterise and compare the glycosylation profiles of five ESA products; Eprex® (epoetin alfa), NeoRecormon® (epoetin beta), Binocrit® (epoetin alfa biosimilar), Silapo (epoetin alfa biosimilar) and Aranesp® (darbepoetin alfa). The methods utilised include mixed-mode anion-exchange/hydrophilic interaction chromatography (AEX/HILIC-MS) for N-glycan identification and quantitation, and HILIC-MS for O-glycan characterisation. The products exhibit notable differences in N- and O-glycosylation, including attributes such as sialic acid occupation, O-acetylation, N-acetyllactosamine extended antennae and sulphated/penta-sialylated N-glycans, which have the potential to cause divergence of therapeutic potencies. The study highlights the need for continued monitoring of ESA product glycosylation, ideally allied to pharmacological data, in order to ensure consistency and therapeutic equivalence between products and enhance our understanding of ESA structure-activity-relationships.
Asunto(s)
Hematínicos/química , Polisacáridos/química , Espectrometría de Masas en Tándem/métodos , Acetilación , Amino Azúcares/química , Técnicas Biosensibles , Cromatografía Líquida de Alta Presión , Darbepoetina alfa/química , Epoetina alfa/química , Eritropoyetina/química , Glicosilación , Estructura Molecular , Ácido N-Acetilneuramínico/química , Proteínas Recombinantes/químicaRESUMEN
In the present study, two conventional (with and without sand filter) and four additional (moving bed biofilm reactor, ozone, moving bed biofilm reactor combined with ozone and a membrane bio reactor) treatment technologies were operated in small-scale at Hammarby Sjöstad sewage treatment plant, Stockholm, Sweden. The effluents were tested with five short-term (≤ 7 days exposure) ecotoxicological tests, and analyzed for a number of target analytes, comprising pharmaceuticals, natural hormones and industrial chemicals. Overall, the tested effluents generated few adverse effects at lower concentrations (< 50% sewage effluent), and no major differences were observed between any of the treatments. The effluent treated with the moving bed biofilm reactor resulted in the lowest effects in the ecotoxicological tests. The most efficient treatment technology with regard to the pharmaceutical residues was the ozone treatment, which however caused negative effects in some of the ecotoxicological tests.
Asunto(s)
Reactores Biológicos , Residuos de Medicamentos/análisis , Disruptores Endocrinos/análisis , Hormonas/análisis , Aguas del Alcantarillado/química , Eliminación de Residuos Líquidos/métodos , Contaminantes Químicos del Agua/análisis , Análisis de Varianza , Animales , Chlorophyta/efectos de los fármacos , Crustáceos/efectos de los fármacos , Residuos de Medicamentos/toxicidad , Embrión no Mamífero/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Hormonas/toxicidad , Ozono/metabolismo , Rhodophyta/efectos de los fármacos , Suecia , Pruebas de Toxicidad , Vibrio/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Pez Cebra/metabolismoRESUMEN
A novel solid-phase extraction (SPE) method is presented whereby 15 basic, neutral and acidic pharmaceuticals in wastewater were simultaneously extracted and subsequently separated into different fractions. This was achieved using mixed-mode cation- and anion-exchange SPE (Oasis MCX and MAX) in series. Analysis was performed by high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC/QTOF-MS). A fast separation was achieved, with all compounds eluting within 6min, narrow chromatographic peaks, with a peak base width of 6s on average, and a high mass accuracy of quantified wastewater sample ions, with average mass errors in absolute value of 0.7mDa or 2.7ppm. The recovery of the SPE method in the analysis of sewage treatment plant (STP) influent and effluent wastewater was on average 80% and the ion suppression 30%. For less demanding samples Oasis MCX used alone may be an alternative method, although for STP influent waters containing high loads of organic compounds the clean-up achieved using only Oasis MCX was insufficient, leading to unreliable quantitation. Furthermore, serial SPE separation according to molecular charge added an additional degree of analyte confirmation. For quantitation, an approach combining external standard calibration curves, isotopically labelled surrogate standards and single-point standard addition was used. The applicability of the method was demonstrated in the analysis of influent and effluent wastewater from an STP, using small sample volumes (25-50mL). The effluent wastewater had been subjected to three different treatments; activated sludge, activated sludge followed by ozonation, and a membrane bioreactor (MBR). Ozone treatment proved superior in removal of the analysed pharmaceuticals, while the MBR provided higher removal efficiencies than the activated sludge process.
Asunto(s)
Preparaciones Farmacéuticas/aislamiento & purificación , Aguas del Alcantarillado/química , Extracción en Fase Sólida/métodos , Eliminación de Residuos Líquidos , Contaminantes Químicos del Agua/aislamiento & purificación , Reactores Biológicos , Cromatografía Líquida de Alta Presión , Monitoreo del Ambiente , Espectrometría de Masas , Ozono , Preparaciones Farmacéuticas/química , Solventes/químicaRESUMEN
A method for studying peptide-surface interactions within microfluidic channels by radionuclide imaging is described. With the high surface area-to-volume ratio of channels in miniaturised devices, combined with low amounts of analyte, non-specific peptide adsorption is a critical issue. The objective of the study was therefore to develop a method capable of direct detection of adsorbed peptide within microfluidic channels. A micro-device consisting of channels moulded in a plastic compact disc was chosen for the study, together with two selected peptides of different lengths and isoelectric point (pI) values. A bifunctional chelator, DOTA, was attached to the peptide by conjugation and labelled with the short-lived positron emitting radionuclide 68Ga. Quantitative images of radiotracer distribution within the microfluidic channels were obtained using a PhosphorImager system. The power of the method was demonstrated by the ability to clearly measure changes in adsorption when varying a number of parameters that typically affect peptide adsorption. These included surface modifications, analyte concentration, pH, and ionic strength. Additionally, two quantification methods were developed and compared. Radionuclide imaging also permitted visualisation of adsorption and release processes in microchannel chromatographic columns. The results suggest that radionuclide imaging is a suitable tool not only for the study of peptide adsorption to the microchannels presented in this study but also as a versatile tool to measure peptide-surface interactions in a wide variety of miniaturised structures and devices.
Asunto(s)
Radioisótopos de Galio/química , Técnicas Analíticas Microfluídicas/métodos , Péptidos/análisis , Plásticos/química , Adsorción , Diseño de Equipo , Técnicas Analíticas Microfluídicas/instrumentación , Propiedades de SuperficieRESUMEN
The poly(dimethylsiloxane) (PDMS) material provides a number of advantageous features, such as flexibility, elasticity, and transparency, making it useful in integrated analytical systems. Hard fused-silica capillary structures and soft PDMS channels can easily be combined by a tight fit, which offers many alternatives for structure combinations. PDMS and fused silica are in different ways prone to adsorption of low levels of organic compounds. The need for modification of the inner wall surface of PDMS channels may often be necessary, and in this paper, we describe an easy and effective method using the amine-containing polymer PolyE-323 to deactivate both fused-silica and PDMS surfaces. The adsorption of selected peptides to untreated surfaces was compared to PolyE-323-modified surfaces, using both radionuclide imaging and capillary electrophoresis experiments. The polyamine modification displayed a substantially reduced adsorption of three hydrophobic test peptides compared to the native PDMS surface. Filling and storage of aqueous solution were also possible in PolyE-323-modified PDMS channels. In addition, hybrid microstructures of fused silica and PDMS could simultaneously be deactivated in one simple coating procedure.
Asunto(s)
Dimetilpolisiloxanos/química , Electroforesis Capilar/métodos , Poliaminas/química , Cintigrafía/métodos , Adsorción , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/análisis , Dióxido de Silicio/químicaRESUMEN
We propose radionuclide imaging as a valuable tool for the study of molecular interactions in miniaturized systems for chemical analysis. Sensitive and quantitative imaging can be performed with compounds labeled with short-lived positron-emitting radionuclides, such as (11)C and (68)Ga, within selected parts of the system. Radionuclide imaging is not restricted to transparent materials since the relatively energetic positrons can penetrate high optical density materials. Experimentally, a radiotracer is introduced into the object of study, which is subsequently placed on a phosphor storage plate. After exposure, the plate is scanned with a laser and a digital, quantitative image can be reconstituted. To demonstrate the concept, three types of microstructures suited for integration in chemical analysis systems were imaged with (11)C- and (68)Ga-labeled tracers. The influence of factors such as geometry of the object and type of radionuclide on resolution and sensitivity was investigated. The resolution ranged from 0.9 to 2.7 mm (fwhm). Measuring low amounts of radioactivity in the three structures, 2-20 Bq could be detected, which corresponded to 2.3-500 amol or 2.4-110 pM tracer. The imaging approach was applied to study analyte concentration and sample dilution effects on the performance of a capillary extraction column integrated in an automated LC-ESI-MS system. The utility of the technique was further illustrated by imaging of microchannels in a zeonor plastic compact disk and in a poly(dimethylsiloxane) material for the study of nonspecific peptide adsorption.