Selective ionization of oxidized lipid species using different solvent additives in flow injection mass spectrometry.

Lipid oxidation in food products is a crucial problem that causes undesirable changes in the food's flavor, texture, and nutritional value. It should be carefully monitored as it can lead to the formation of potentially toxic compounds and in that way reduce the shelf life of the product. Liquid chromatography coupled to mass spectrometry is a powerful tool to monitor the formation of oxidized lipids. However, the presence of lipid species in both their non-oxidized and oxidized forms at distinctly different concentrations can hinder the detection and identification of the less abundant oxidized species, due to coelution. In this study, a flow injection mass spectrometry approach was used to selectively ionize oxidized triacylglycerols versus their non-oxidized precursors. Three mobile phase additives were investigated (ammonium formate, sodium acetate, and sodium iodide) at three different concentrations, and ion source settings (i.e., sheath gas temperature, capillary voltage, and nozzle voltage) were optimized. A fractional factorial design was conducted to examine not only the direct effect of the operating parameters on the selectivity of ionization for the oxidized lipid species, but also to assess their combined effect. Overall, selective ionization of oxidized versus non-oxidized lipid species was favored by the use of sodium-containing solvent additives. The application of specific ion source settings resulted in an increased ionization selectivity, with sheath gas temperature and capillary voltage having the most significant influence. A selectivity factor as high as 120 could be reached by combining 0.1 mg/mL sodium-containing additives, with 250 °C sheath gas temperature and 5000 V capillary voltage. These findings will contribute to future studies on fast detection and relative quantification of low abundant oxidized triacylglycerols and their possible impact on human health.

Co-expression of squalene epoxidases with triterpene cyclases boosts production of triterpenoids in plants and yeast.

Triterpene cyclases catalyze the first committed step in triterpene biosynthesis, by forming mono- to pentacyclic backbone structures from oxygenated C30 isoprenoid precursors. Squalene epoxidase precedes this cyclization by providing the oxygenated and activated substrate for triterpene biosynthesis. Three squalene epoxidases from Cucurbita pepo (CpSEs) were isolated and shown to have evolved under purifying selection with signs of sites under positive selection in their N- and C-termini. They all localize to the Endoplasmic Reticulum (ER) and produce 2,3-oxidosqualene and 2,3:22,23-dioxidosqualene when expressed in a yeast erg1 (squalene epoxidase) erg7 (lanosterol synthase) double mutant. Co-expression of the CpSEs with four different triterpene cyclases, either transiently in Nicotiana benthamiana or constitutively in yeast, showed that CpSEs boost triterpene production. CpSE2 was the best performing in this regard, which could reflect either increased substrate production or superior channeling of the substrate to the triterpene cyclases. Fluorescence Lifetime Imaging Microscopy (FLIM) analysis with C. pepo cucurbitadienol synthase (CpCPQ) revealed a specific interaction with CpSE2 but not with the other CpSEs. When CpSE2 was transformed into C. pepo hairy root lines, cucurbitacin E production was increased two folds compared to empty vector control lines. This study provides new insight into the importance of SEs in triterpene biosynthesis, suggesting that they may facilitate substrate channeling, and demonstrates that SE overexpression is a new tool for increasing triterpene production in plants and yeast.

A comprehensive two-dimensional liquid chromatography method for the simultaneous separation of lipid species and their oxidation products.

Lipid oxidation is one of the major causes of food spoilage for lipid-rich foods. In particular, oil-in-water emulsions, like mayonnaises and spreads, are prone to oxidation due to the increased interfacial area that facilitates contact between the lipids and hydrophilic pro-oxidants present in the water phase. Polar, amphiphilic lipid species present at the oil/water interface, like the mono- (MAGs) and di-acylglycerols (DAGs), act as oxidation starters that initiate subsequent oxidation reactions of the non-polar lipids in the oil droplets. A comprehensive two-dimensional liquid chromatography (LC×LC) method with evaporative light-scattering detection (ELSD) was set up to study the composition of the complex mixture of oxidized polar and non-polar lipids. The LC×LC-ELSD method employs size exclusion chromatography (SEC) in the 1D (1st dimension) to separate the various lipid species according to size. In the 2D (2nd dimension), normal-phase liquid chromatography (NPLC) is used to separate the fractions according to their degree of oxidation. The coupling of SEC with NPLC yields a good separation of the oxidized triacylglycerols (TAGs) from the large excess of non-oxidized TAGs. In addition, it allows the isolation of non-oxidized DAGs and MAGs that usually interfere with the detection of a variety of oxidized products that have similar polarities. This method facilitates elucidating how lipid composition affects oxidation kinetics in emulsified foods and will aid in the development of more oxidation-stable products.

Glucosinolate turnover in Brassicales species to an oxazolidin-2-one, formed via the 2-thione and without formation of thioamide.

Glucosinolates are found in plants of the order Brassicales and hydrolyzed to different breakdown products, particularly after tissue damage. In Barbarea vulgaris R.Br. (Brassicaceae), the dominant glucosinolate in the investigated "G-type" is glucobarbarin, (S)-2-hydroxy-2-phenylethylglucosinolate. Formation of the nitrile from glucobarbarin was observed in vitro, while a previously suggested thioamide (synonym thionamide) was not confirmed. Resedine (5-phenyl-1,3-oxazolidin-2-one) was detected after glucobarbarin hydrolysis in crushed B. vulgaris leaves and siliques, but not in intact parts. The abundance increased for several hours after completion of hydrolysis. The corresponding 1,3-oxazolidine-2-thione (OAT), with the common name barbarin, was also formed, and appeared to be the precursor of resedine. Addition of each of two non-endogenous OATs, (S)-5-ethyl-5-methylOAT and (R)-5-vinylOAT (R-goitrin), to a leaf homogenate resulted in formation of the corresponding 1,3-oxazolidin-2-ones (OAOs), confirming the metabolic connection of OAT to OAO. Formation of OAOs was inhibited by prior brief heating of the homogenate, suggesting enzyme involvement. We suggest the conversion of OATs to OAOs to be catalyzed by an enzyme ("oxazolidinethionase") responsible for turnover of OAT formed in intact plants. Resedine had been reported as an alkaloid from another species - Reseda luteola L. (Resedaceae) - naturally containing the glucosinolate glucobarbarin. However, resedine was not detected in intact R. luteola plants, but formed after tissue damage. The formation of resedine in two families suggests a broad distribution of putative OATases in the Brassicales; potentially involved in glucosinolate turnover that needs myrosinase activity as the committed step. In agreement with the proposed function of OATase, several candidate genes for myrosinases in glucosinolate turnover in intact plants were discovered in the B. vulgaris genome. We also suggest that biotechnological conversion of OATs to OAOs might improve the nutritional value of Brassicales protein. HPLC-MS/MS methods for detection of these glucobarbarin products are described.