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1.
J Chromatogr A ; 1605: 360355, 2019 Nov 08.
Article in English | MEDLINE | ID: mdl-31315811

ABSTRACT

Urea, as an end product of protein metabolism and an abundant polar compound, significantly complicates the metabolomic analysis of urine by GC-MS. We developed a sample preparation method removing urea from urine samples prior the GC-MS analysis. The method based on urease immobilized on magnetic microparticles was compared with the others that are conventionally used (liquid-liquid extraction, free urease protocol), and samples without any treatment. To study the impact of sample preparation approaches on the quality of analytical data, we employed comprehensive metabolomic analysis (using both GC-MS and LC-MS/MS platforms) of standard material based on human urine. Multivariate statistical analysis has shown that immobilized urease treatment provides similar results to a free urease approach. However, significant alterations in the profiles of metabolites were observed in the samples without any treatment and after the extraction. Compared to other approaches that were tested, the immobilization of urease on microparticles reduces both the number of artifacts and the variability of the metabolites (average CV of extraction 19.7%, no treatment 11.4%, free urease 5.0%, and immobilized urease 2.5%). The method that was developed was applied in a GC-MS metabolomic experiment of glutaric aciduria type I, where both known diagnostically important biomarkers and unknowns, as the most discriminating compounds, were found.


Subject(s)
Analytic Sample Preparation Methods , Enzymes, Immobilized/urine , Gas Chromatography-Mass Spectrometry/methods , Magnetic Phenomena , Metabolomics/methods , Urease/urine , Amino Acid Metabolism, Inborn Errors/metabolism , Brain Diseases, Metabolic/metabolism , Chromatography, Liquid/methods , Feasibility Studies , Glutaryl-CoA Dehydrogenase/deficiency , Glutaryl-CoA Dehydrogenase/metabolism , Humans , Metabolome , Principal Component Analysis , Reproducibility of Results , Tandem Mass Spectrometry , Urea/metabolism
2.
Biomaterials ; 30(15): 2855-63, 2009 May.
Article in English | MEDLINE | ID: mdl-19264355

ABSTRACT

Nearly monodispersed superparamagnetic maghemite nanoparticles (15-20nm) were prepared by a one-step thermal decomposition of iron(II) acetate in air at 400 degrees C. The presented synthetic route is simple, cost effective and allows to prepare the high-quality superparamagnetic particles in a large scale. The as-prepared particles were exploited for the development of magnetic nanocomposites with the possible applicability in medicine and biochemistry. For the purposes of the MRI diagnostics, the maghemite particles were simply dispersed in the bentonite matrix. The resulting nanocomposite represents very effective and cheap oral negative contrast agent for MRI of the gastrointestinal tract and reveals excellent contrast properties, fully comparable with those obtained for commercial contrast material. The results of the clinical research of this maghemite-bentonite contrast agent for imaging of the small bowel are discussed. For biochemical applications, the primary functionalization of the prepared maghemite nanoparticles with chitosan was performed. In this way, a highly efficient magnetic carrier for protein immobilization was obtained as demonstrated by conjugating thermostable raffinose-modified trypsin (RMT) using glutaraldehyde. The covalent conjugation resulted in a further increase in trypsin thermostability (T(50)=61 degrees C) and elimination of its autolysis. Consequently, the immobilization of RMT allowed fast in-solution digestion of proteins and their identification by MALDI-TOF mass spectrometry.


Subject(s)
Contrast Media , Enzymes, Immobilized , Ferric Compounds , Magnetic Resonance Imaging , Trypsin , Gastrointestinal Tract/pathology , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , X-Ray Diffraction
3.
J Enzyme Inhib Med Chem ; 20(3): 261-7, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16119197

ABSTRACT

Inhibition of porcine pancreas and human saliva alpha-amylase (EC 3.2.1.1) by sanguinarine and chelerythrine was studied. The inhibition of alpha-amylase was assayed using a biosensor method which utilises a flow system equipped with a peroxide electrode. 250 microM sanguinarine and 250 microM chelerythrine cause complete inhibition of 1.9 nkat alpha-amylase from porcine pancreas. The same concentration of sanguinarine and chelerythrine caused 23.9% and 7.5% inhibition, respectively, of 1.9 nkat alpha-amylase from human saliva. Mixed type and partially reversible inhibition was found for both alpha-amylases treated with either alkaloid.


Subject(s)
Alkaloids/metabolism , Phenanthridines/metabolism , alpha-Amylases/antagonists & inhibitors , Alkaloids/pharmacology , Animals , Benzophenanthridines , Biosensing Techniques , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Enzyme Stability , Humans , Isoquinolines , Kinetics , Phenanthridines/pharmacology , Swine , Time Factors , alpha-Amylases/metabolism
4.
Biosens Bioelectron ; 20(2): 240-5, 2004 Sep 15.
Article in English | MEDLINE | ID: mdl-15308227

ABSTRACT

A new biosensing flow injection method for the determination of alpha-amylase activity has been introduced. The method is based on the analysis of maltose produced during the hydrolysis of starch in the presence of alpha-amylase. Maltose determination in the flow system was allowed by the application of peroxide electrode equipped with an enzyme membrane. The membrane was obtained by immobilisation of glucose oxidase, alpha-glucosidase and optionally mutarotase on a cellophane, co-crosslinked by gelatin-glutaraldehyde together with bovine serum albumine. alpha-Glucosidase hydrolyses maltose to alpha-D-glucose, which is converted to beta-D-glucose by mutarotase. beta-D-Glucose is then determined via glucose oxidase. The new biosensor has the limit of detection of 50 nmol l(-1) maltose, which means 2 nkat ml(-1) in alpha-amylase activity units, when the reaction time of amylase was 5 min (determined with respect to a signal-to-noise ratio 3:1). When the reaction time of alpha-amylase was 30 min, the limit of detection was 0.5 nkat ml(-1). A linear range of current response was 0.1-3 mmol l(-1) maltose, with a response time of 35s. The biosensor was stable at least two months and retained 70% of its original activity (with mutarotase the stability is decreased to 3 weeks). When the enzyme membrane was stored in a dry state at 4 degrees C in a refrigerator, the lifetime was approximately 6 months (with mutarotase only 3 months).


Subject(s)
Biosensing Techniques/instrumentation , Electrochemistry/instrumentation , Flow Injection Analysis/instrumentation , Maltose/analysis , Maltose/chemistry , alpha-Amylases/analysis , alpha-Amylases/chemistry , Biosensing Techniques/methods , Carbohydrate Epimerases/chemistry , Electrochemistry/methods , Enzyme Activation , Enzymes, Immobilized/chemistry , Equipment Design , Equipment Failure Analysis , Flow Injection Analysis/methods , Glucose Oxidase/chemistry , Reproducibility of Results , Sensitivity and Specificity , alpha-Glucosidases/chemistry
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