The use of a liquid chromatography (LC) splitter inserted between an HPLC column and an evaporative light scattering detector (ELSD) is described. This paper aims to show the feasibility of using the splitter in an HPLC-ELSD system to fractionate a model mixture of analytes, namely salicin (2-(hydroxymethyl)-phenyl-β-d-glucopyranoside) and glucose. The retention factors and efficiency of the separation were studied under various temperatures and water contents in the mobile phase in order to clarify the mechanism of polyols separation on a diol column under the conditions of hydrophilic liquid chromatography (HILIC). Finally, the system was applied to a biological sample (a lyophilized colony gel of Pectinatella magnifica), where the presence of fructose and glucose was confirmed.
Puerarin-7-O-fructoside was transformed by Trichoderma harzianum CGMCC 1523 into 3'-hydroxypuerarin-7-O-fructoside; this was identified by MS and NMR. However, puerarin-7-O-glucoside was not directly hydroxylated but hydrolyzed back into puerarin, which was transformed into 3'-hydroxypuerarin by the same fungi. Comparative analysis of free radical scavenging activity of DPPH showed that the free radical scavenging activity of puerarin-7-O-glucoside was reduced to approximately 1/2 of that of puerarin, while the free radical scavenging activity of puerarin-7-O-fructoside was increased to approximately 1.5 times of that of puerarin. The free radical scavenging activity of 3'-hydroxypuerarin-7-O-fructoside was further increased by 2.2 times of that of puerarin-7-O-fructoside, which was close to that of 3'-hydroxypuerarin.
Glycation is a process closely related to the aging and pathogenesis of diabetic complications. In this process, reactive α-dicarbonyl compounds (e.g., methylglyoxal) cause protein modification accompanied with potential loss of their biological activity and persistence of damaged molecules in tissues. We suppose that glutathione S-transferases (GSTs), a group of cytosolic biotransformation enzymes, may be modified by glycation in vivo, which would provide a rationale of its use as a model protein for studying glycation reactions. Glycation of GST by methylglyoxal, fructose, and glucose in vitro was studied. The course of protein glycation was evaluated using the following criteria: enzyme activity, formation of advanced glycation end-products using fluorescence and western blotting, amine content, protein conformation, cross linking and aggregation, and changes in molecular charge of GST. The ongoing glycation by methylglyoxal 2 mM resulted in pronounced decrease in the GST activity. It also led to the loss of 14 primary amino groups, which was accompanied by changes in protein mobility during native polyacrylamide gel electrophoresis. Formation of cross links with molecular weight of 75 kDa was observed. Obtained results can contribute to understanding of changes, which proceed in metabolism of xenobiotics during diabetes mellitus and ageing.
- MeSH
- diabetes mellitus enzymologie MeSH
- fruktosa chemie metabolismus MeSH
- glukosa chemie metabolismus MeSH
- glutathiontransferasa chemie metabolismus MeSH
- katalýza MeSH
- lidé MeSH
- produkty pokročilé glykace chemie metabolismus MeSH
- pyruvaldehyd chemie metabolismus MeSH
- stárnutí metabolismus MeSH
- xenobiotika chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The application of an internal standard in quantitative analysis is desirable in order to correct for variations in sample preparation and instrumental response. In mass spectrometry of organic compounds, the internal standard is preferably labelled with a stable isotope, such as (18)O, (15)N or (13)C. In this study, a method for the quantification of fructo-oligosaccharides using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF MS) was proposed and tested on raftilose, a partially hydrolysed inulin with a degree of polymeration 2-7. A tetraoligosaccharide nystose, which is chemically identical to the raftilose tetramer, was used as an internal standard rather than an isotope-labelled analyte. Two mathematical approaches used for data processing, conventional calculations and artificial neural networks (ANN), were compared. The conventional data processing relies on the assumption that a constant oligomer dispersion profile will change after the addition of the internal standard and some simple numerical calculations. On the other hand, ANN was found to compensate for a non-linear MALDI response and variations in the oligomer dispersion profile with raftilose concentration. As a result, the application of ANN led to lower quantification errors and excellent day-to-day repeatability compared to the conventional data analysis. The developed method is feasible for MS quantification of raftilose in the range of 10-750 pg with errors below 7%. The content of raftilose was determined in dietary cream; application can be extended to other similar polymers. It should be stressed that no special optimisation of the MALDI process was carried out. A common MALDI matrix and sample preparation were used and only the basic parameters, such as sampling and laser energy, were optimised prior to quantification.
- MeSH
- fakoemulzifikace využití MeSH
- finanční podpora výzkumu jako téma MeSH
- fluorescence MeSH
- fruktosa chemie MeSH
- katarakta patofyziologie MeSH
- lidé MeSH
- oční čočka chemie MeSH
- oxidační stres genetika účinky záření MeSH
- produkty pokročilé glykace MeSH
- studie případů a kontrol MeSH
- transaminasy diagnostické užití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- techniky in vitro MeSH
In an in vitro model with purified porcine aspartate aminotransferase (AST, EC 2.6.1.1) as the protein, the effects of phenolic antioxidants of plant origin (arbutin, methylarbutin, ferulic and isoferulic acids, o-coumaric and p-coumaric acids, quinic acid), flavonoids (baicalin and baicalein), and of hydroxycitric acid (HCA) at 0.5-50 mM concentration on the enzyme activities and on its glycation by 50 mM D-fructose as the glycating agent were studied. During incubation with AST at 37 degrees C up to 24 days, fructose alone decreased AST activities as a result of protein glycation. In the absence of fructose, 50 mM phenolic compounds gradually decreased AST activity, while no or a weak effect of individual compounds was found at 3 mM concentration. A direct negative effect on AST was pronounced with ferulic acid. On the other hand, beneficial influences of phenolic compounds on glycation of AST by fructose were found mostly at 3 mM concentration. Effects on glycation were vague at 50 mM concentration, probably due to a combination of direct negative influences and antiglycation effects of individual compounds. No effect, neither positive nor negative, on AST activity and protein glycation, was found with quinic acid. The flavonoid baicalin and its aglycon baicalein rapidly decreased the in vitro activity of the enzyme in all concentrations used (0.5-3 mM), and no beneficial effects of the compounds on glycation of the enzyme by fructose were found. The influence of HCA on glycation was concentration-depended, ranging from beneficial inhibition of glycation at 2.5 mM concentration to a strong decrease in AST activity at 10 mM HCA. Both the beneficial and undesirable effects of natural antioxidants should be considered in case they are used as antiglycation factors. The results obtained can contribute to the evaluation of quality of various generally recommended antioxidants.
- MeSH
- antioxidancia analýza chemie MeSH
- Arctostaphylos MeSH
- biologické přípravky analýza chemie MeSH
- financování organizované MeSH
- fruktosa analýza chemie MeSH
- listy rostlin MeSH
- oxidace-redukce MeSH
- prasata MeSH
- proteiny analýza chemie MeSH
- rostlinné extrakty analýza chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- hodnotící studie MeSH
