Targeted Metabolomics: The LC-MS/MS Based Quantification of the Metabolites Involved in the Methylation Biochemical Pathways.

Biochemical methylation reactions mediate the transfer of the methyl group regulating vital biochemical reactions implicated in various diseases as well as the methylation of DNA regulating the replication processes occurring in living organisms. As a finite number of methyl carriers are involved in the methyl transfer, their quantification could aid towards the assessment of an organism's methylation potential. An Hydrophilic Interaction Chromatography-Liquid Chromatography Multiple Reaction Monitoring (HILIC-LC-MRM) mass spectrometry (MS) methodology was developed and validated according to Food & Drug Administration (FDA), European Medicines Agency (EMA), and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) for the simultaneous determination of nine metabolites i.e., B12, folic acid, 5-methyltetrahydrofolate, S-adenosylmethionine, S-adenosylhomocysteine, betaine, phosphocholine, N,N-dimethylglycine, and deoxythymidine monophosphate in human blood plasma. The sample pretreatment was based on a single step Solid-phase extraction (SPE) methodology using C18 cartridges. The methodology was found to accurately quantitate the analytes under investigation according to the corresponding dynamic range proposed in the literature for each analyte. The applicability of the method was assessed using blood donor samples and its applicability demonstrated by the assessment of their basal levels, which were shown to agree with the established basal levels. The methodology can be used for diagnostic purposes as well as for epigenetic screening.

Evolved Gas Analysis-Mass Spectrometry to Identify the Earliest Organic Binder in Aegean Style Wall Paintings.

An organic binder was identified in the painted fragments from the Canaanite palace of Tel Kabri, Israel. Recently dated to the late 18th century B.C.E. by 14 C, Tel Kabri is the most ancient of the Eastern Mediterranean sites in which Aegean style paintings have been found. The application of pigments was suspected to be using an organic binding medium, particularly for the Egyptian Blue pigment. Samples of blue paint were examined using evolved gas analysis-mass spectrometry (EGA-MS) in order to overcome the analytical challenges imposed by highly degraded aged proteinaceous materials. Egg was identified as the binder based on the presence of hexadecanonitrile and octadecanonitrile, confirming the use of a secco painting technique. Lysozyme C from Gallus gallus was detected by proteomics analysis, confirming the presence of egg. To our knowledge, this is the earliest use of egg as a binder in Aegean style wall paintings.

Roasted and green coffee extracts show antioxidant and cytotoxic activity in myoblast and endothelial cell lines in a cell specific manner.

Coffee is one of the most highly consumed beverages with potential beneficial health implications, however its molecular mechanism of action has not been completely elucidated yet. To that cause, the polyphenolic composition of different coffee extracts (from Light, Medium and Dark roasts as well as green beans) was examined by UHPLC-HRMS analysis, indicating chlorogenic acids isomers as the main constituents. In the following step, the toxicity of the extracts was tested in myoblasts and endothelial cells and differential toxicity of green and roasted samples was displayed as the myoblasts were more sensitive to green coffee extracts, in contrast to the endothelial cells. Subsequently, biologically relevant, non-cytotoxic extract concentrations were administered to explore their potential effect on cell redox status using flow cytometry and spectrophotometric assays. The results indicated that all coffee extracts improved cell redox status, however differences were observed between the two different cell lines tested, implying that coffee compounds display cell- and tissue-specificity. Glutathione levels were increased in almost all cases up to 70%, while the roasting degree affected the free radical scavenging potential of the extracts and their ability to protect from macromolecular oxidation as exhibited by the differences in ROS, CARB and TBARS levels, especially in the myoblasts.

Effect of polyphenols from coffee and grape on gene expression in myoblasts.

Coffee and grape contain various bioactive compounds like polyphenols that may exert beneficial effects, especially antioxidant activity, on human health upon consumption. However, the molecular mechanisms through which these effects are achieved are not fully elucidated. Thus, in the present study in order to investigate these mechanisms, a whole genome expression DNA microarray analysis was carried out in myoblasts treated with polyphenols of coffee and grape pomace at concentrations that improved the redox status. Grape was composed of catechin, epicatechin, cyanidin, malvidin, delphinidin, petunidin, myrtillin, kuromanin, oenin, peonidin, quercetin, gallic acid and caftaric acid as LC-MS revealed, with a total polyphenolic content (TPC) of 648 mg of gallic acid equivalents/g of dry matter. Coffee had a TPC of 42.61 mg GAE/g coffee and was composed of 3-chlorogenic acid (16.61 mg/g), 4- and 5-chlorogenic acids (13.62 mg/g), as UHPLC-HRMS revealed. According to the results, grape polyphenols altered mainly the expression of cytoskeleton and differentiation-associated genes, while coffee compounds had a more profound effect, on the expression levels of many metabolic and antioxidant genes possibly through the nuclear factor (erythroid-derived 2) like-2 (Nrf2) pathway.

Enhancement of Antioxidant Mechanisms and Reduction of Oxidative Stress in Chickens after the Administration of Drinking Water Enriched with Polyphenolic Powder from Olive Mill Waste Waters.

The aim of the study was to examine the effects of a polyphenolic powder from olive mill wastewater (OMWW) administered through drinking water, on chickens' redox status. Thus, 75 chickens were divided into three groups. Group A was given just drinking water, while groups B and C were given drinking water containing 20 and 50 µg/ml of polyphenols, respectively, for 45 days. The antioxidant effects of the polyphenolic powder were assessed by measuring oxidative stress biomarkers in blood after 25 and 45 days of treatment. These markers were total antioxidant capacity (TAC), protein carbonyls (CARB), thiobarbituric acid reactive species (TBARS) and superoxide dismutase activity (SOD) in plasma, and glutathione (GSH) and catalase activity in erythrocytes. The results showed that CARB and TBARS were decreased significantly in groups B and C, and SOD decreased in group B compared to that in group A. TAC was increased significantly in group C and GSH was increased in group B, while catalase activity was increased in groups B and C compared to that in group A. In conclusion, this is the first study showing that supplementation of chickens with polyphenols from OMWW through drinking water enhanced their antioxidant mechanisms and reduced oxidative stress-induced damage.