Evaluation of Saliva Stability for NMR Metabolomics: Collection and Handling Protocols

Maintaining a salivary metabolic profile upon sample collection and preparation is determinant in metabolomics. Nuclear magnetic resonance (NMR) spectroscopy was used to identify metabolite changes during short-term storage, at room temperature (RT)/4 °C/−20 °C, and after sample preparation, at RT/4 °C (mimicking typical clinical/laboratory settings). Interestingly, significant metabolic inter-individual and inter-day variability were noted, probably determining sample stability to some extent. After collection, no changes were noted at −20 °C (at least for 4 weeks). RT storage induced decreases in methylated macromolecules (6 h); lactate (8 h); alanine (12 h); galactose, hypoxanthine, pyruvate (24 h); sarcosine, betaine, choline, N-acetyl-glycoproteins (48 h), while acetate increased (48 h). Less, but different, changes were observed at 4 °C, suggesting different oral and microbial status at different temperatures (with a possible contribution from inter-individual and inter-day variability), and identifying galactose, hypoxanthine, and possibly, choline esters, as potential general stability indicators. After preparation, addition of NaN3 did not impact significantly on saliva stabilization, neither at RT nor at 4 °C, although its absence was accompanied by slight increases in fucose (6.5 h) and proline (8 h) at RT, and in xylose (24 h) at 4 °C. The putative metabolic origins of the above variations are discussed, with basis on the salivary microbiome. In summary, after collection, saliva can be stored at RT/4 °C for up to 6 h and at −20 °C for at least 4 weeks. Upon preparation for NMR analysis, samples are highly stable at 25 °C up to 8 h and at 4 °C up to 48 h, with NaN3 addition preventing possible early changes in fucose, proline (6–8 h), and xylose (24 h) levels.

The identification of acetylcholine and choline in oat seedlings by gas chromatography and nuclear magnetic resonance (NMR)

Four methods of isolation and purification of choline esters from green 7-day-old oat ( <em>Avena sativa</em> L. cv. Diadem) seedlings were tested The results showed that the best recovery of acetylcholine and choline from plant tissues was obtained using an extraction solution composed of 15% 1N formic acid and 85%, acetone followed by precipitation of both these substances with ammonium reineckate. The presence of acetylcholine and choline in the plant extracts was confirmed by nuclear magnetic resonance (NMR) and gas chromatography. In the case of gas chromatography, after isolation and purification of the studied compounds from the plant material, estrification of choline followed by N-demethylation of acetylcholine and estrified choline were performed The demethylation reaction was conducted in a reaction mixture of 50 mM sodium thiophenolate and 25 mM thiophenol in anhydrous acetone. After its completion, the mixture was removed with pentanone and the demethylated esters were extracted into chloroform.

Correlation between butyrylcholinesterase variants and sensitivity to soman toxicity.

Butyrylcholinesterase (BChE) is synthesized in the liver and found in high concentrations in blood plasma, liver, heart, pancreas, vascular endothelium, skin, brain white matter, smooth muscle cells and adipocytes. BChE is a non specific enzyme that hydrolyzes different choline esters (succinylcholine, mivacurium) and many other drugs such as aspirin, cocaine and procaine. The enzyme is also considered as a bioscavenger due to its ability to neutralize the toxic effects of organophosphorus compounds (nervous system fs agents) such as soman. BChE displays several polymorphisms that influence its serum activity; therefore they could determine the individual sensitivity to chemical nerve agents. In this study, we investigated the correlation between BChE variants and the degree of enzyme inhibition and reactivation after soman application on blood samples of 726 individuals. The blood samples of individuals expressing abnormal variants, were more sensitive to soman compared to variants of homozygotes and heterozygotes for U-allele. We found significant differences in the degree of enzyme reactivation between different variants (with and without U-presence).