Simultaneous determination of genomic DNA methylation and uracil misincorporation.

OBJECTIVE: To develop a method for the simultaneous measurement of 5-methylcytosine (5-metC) and 2'-deoxyuridine monophosphate (dU). MATERIALS AND METHODS: Genomic DNA was extracted from the HepG2 cell line grown in experimental complete medium or in folate-depleted medium. Samples were treated with RNAse A and RNAse T1 to avoid any RNA contamination. High-performance liquid chromatography (HPLC)/electrospray ionization mass spectrometric (ESI-MS) method was used to separate nucleotides after enzymatic hydrolysis of DNA with nuclease P1, phosphodiesterase I and alkaline phosphatase. RESULTS: Using this sensitive new methodology, we were able to quantify simultaneously the concentration of DNA-5-metC and DNA-uracil in DNA. The linear correlation coefficient (R(2)) between the MS signal and the concentration of 5-metC in a range of 0.5-5 microM or dU in a range of 10-100 microM was 0.9954 and 0.9999, respectively. The coefficient of variation was 16.94 and 14.77%, respectively. The applicability of this assay is demonstrated by detection of a decrease in 5-metC% and elevation of dU/thymidylate (dT) into genomic DNA extracted from the HepG2 cell line grown in a folate-depleted medium. CONCLUSION: Our results confirm that the HPLC/ESI-MS method reported earlier for measuring 5-metC allows measurement of uracil misincorporation into DNA.

Isolation and partial characterization of four fluorophores formed by nonenzymatic browning of methylglyoxal and glutamine-derived ammonia.

An aqueous solution of L-glutamine (50 mmol/L) and methylglyoxal (100 mmol/L) was incubated at 120 degrees C for 3 h in a 200 mmol/L phosphate buffer (pH 7.4). Four major fluorophores were revealed on the HPLC chromatogram. The same four fluorophores were obtained from the heating of a mixture of ammonia and methylglyoxal. After purification and concentration, they were structurally characterized by electrospray ionization mass spectrometry (ESI-MS) using the high resolution and tandem mass spectrometry capabilities of a quadrupole time-of-flight MS. The accurate mass measurement of their [M+H]+ ions, the MS fragment patterns, and the presence of one to two nitrogen indicate the formation of fluorophores with molecular formulas of C7H7NO3, C8H9NO3, C12H14N2O4, and C12H14N2O5. These results show that, in an aqueous solution, free glutamine undergoes a rapid degradation, leading to the formation of ammonia which reacts with methylglyoxal to form fluorescent heterocyclic Maillard products.

[The metabolic, nutritional and toxicological consequences of ingested dietary Maillard reaction products: a literature review]. / Etat des connaissances sur la biodisponibilité et la toxicité des produits de Maillard issus de l'alimentation.

The field of Maillard reaction in food has recently re-emerged. This reaction which takes place between carbohydrates and proteins at a high cooking temperatures and causes the formation of flavor and yellow to brown colors was already well documented. Little is known, however, about the formation of other Maillard reaction products (MRPs) which may be toxic: the so-called glycotoxins. It is well recognized that only 10% of these have been identified so far, and improved analytical methods are needed for the discovery of more of the neo-formed contaminants. Only a few studies as yet have focused on the digestion, metabolism and excretion of fructoselysine, carboxymethyllysine, pentosidine, acrylamide, the MRPs which have already been identified. MRPs have been shown to be present at significant amounts in a variety of industrially and domestically heat-treated foodstuffs but their absorption appears to be limited and they are readily excreted. Clinical studies indicate, none the less, that the typical Western diet, which contains a high MRPs content, may have an impact on human health. The main effects are observed on the glucose and lipid metabolisms, and on inflammatory mediators. However, the physiopathological role of the ingested MRPs has yet to be investigated in detail, so no conclusive recommendations can be given at present regarding their possible toxic effects.

In-capillary non-covalent labeling of insulin and one gastrointestinal peptide for their analyses by capillary electrophoresis with laser-induced fluorescence detection.

The potential of the commercially available dye sypro orange for in-capillary derivatization was evaluated for the detection of insulin and one gastrointestinal peptide (Arg-Arg-gastrin) by capillary electrophoresis with laser induced fluorescence (CE-LIF). The fluorescent emission intensity (lambda(ex) = 488 nm, lambda(em) = 610 nm) of this probe is very low in aqueous medium, and increases strongly in less polar solvent, e.g. methanol. The hydrophobic character of the two analyzed peptides is too low to induce sufficient interaction with the fluorescent probe for good sensitivity when the latter is alone in the background electrolyte. Thus, the potential of several neutral, zwitterionic, cationic and anionic surfactants to favor probe/peptide interactions has been evaluated. It was demonstrated that a borate buffer (pH 8.5) containing tetradecyltrimethylammonium bromide (TTAB) in sub-micellar conditions can be considered as the most suitable buffer for insulin CE-LIF analysis. In addition, the method showed a good linearity between insulin concentration and the peak area of the labeled insulin, allowing quantitative measurements. The sensitivity achieved so far is comparable with that achieved with UV absorption detection, but even at this level it is interesting for microchip analysis, in which fluorescence detection is much more commonly available than UV absorption detection.