Amino acids, betaines and related ammonium compounds in Neapolitan limmo, a Mediterranean sweet lime, also known as lemoncetta Locrese.

BACKGROUND: The so-called 'Neapolitan limmo' or 'lemoncetta Locrese' is an old and now rare Mediterranean sweet lime, similar to lemon but smaller. It is a fruit distinguished from orange, lemon, mandarin, and lime for its sweeter, watery, and non-acidic taste, with a pH between 5.6 and 5.9. No compositional studies are currently available for this citrus fruit. Here we report, for the first time, the distribution in the limmo juice of free amino acids and their main derivatives such as betaines and related ammonium compounds. RESULTS: Seven proteinogenic amino acids (proline, asparagine, serine, aspartic acid, glutamine, alanine, and threonine) and a non-protein amino acid (γ-aminobutyric acid) characterize Neapolitan limmo juice. Proline betaine is the predominant betaine. The data were compared with those of other important citrus juices. CONCLUSION: The specific 'taste quality' of Neapolitan limmo juice can be attributed to its peculiar composition in amino acids. The species-specific presence of the ammonium compound derivatives of the amino acid proline, with proline betaine as the predominant betaine, characterize the non-acidic varieties of Mediterranean sweet lime. Our study constitutes an important step towards the repopulation of this ancient plant and its exploitation in food industry. © 2020 Society of Chemical Industry.

Antioxidant and Anti-Inflammatory Activities of Buffalo Milk δ-Valerobetaine.

δ-Valerobetaine (δVB), a constitutive metabolite of ruminant milk, is produced in the rumen from free dietary Nε- trimethyllysine occurring ubiquitously in vegetable kingdom. The biological role of δVB is poorly known. Here, the antioxidant and anti-inflammatory potential of buffalo milk δVB was tested in vitro during high-glucose (HG)-induced endothelial damage. Results indicated that δVB (0.5 mM) ameliorated the HG cytotoxicity (0.57 ± 0.02 vs 0.41 ± 0.018 O.D. ( P < 0.01). Interestingly, buffalo milk extracts enriched with δVB showed improved significant efficacy in decreasing reactive oxygen species, lipid peroxidation, and cytokine release during HG treatment compared to milk extracts alone ( P < 0.05). It is noteworthy that δVB reduced the HG-activated inflammatory signal by modulating SIRT1 (0.96 ± 0.05 vs 0.85 ± 0.04 AU), SIRT6 (0.82 ± 0.04 vs 0.61 ± 0.03 AU), and NF-κB (0.85 ± 0.03 vs 1.23 ± 0.03 AU) ( P < 0.05). On the whole, our data show the first evidence of δVB efficacy in reducing endothelial oxidative stress and inflammation, suggesting a potential role of this betaine as a novel dietary compound with health-promoting properties.

Carnitine Precursors and Short-Chain Acylcarnitines in Water Buffalo Milk.

Ruminants' milk contains δ-valerobetaine originating from rumen through the transformation of dietary Nε-trimethyllysine. Among ruminant's milk, the occurrence of δ-valerobetaine, along with carnitine precursors and metabolites, has not been investigated in buffalo milk, the second most worldwide consumed milk, well-known for its nutritional value. HPLC-ESI-MS/MS analyses of bulk milk revealed that the Italian Mediterranean buffalo milk contains δ-valerobetaine at levels higher than those in bovine milk. Importantly, we detected also γ-butyrobetaine, the l-carnitine precursor, never described so far in any milk. Of interest, buffalo milk shows higher levels of acetylcarnitine, propionylcarnitine, butyrylcarnitine, isobutyrylcarnitine, and 3-methylbutyrylcarnitine (isovalerylcarnitine) than cow milk. Moreover, buffalo milk shows isobutyrylcarnitine and butyrylcarnitine at a 1-to-1 molar ratio, while in cow's milk this ratio is 5 to 1. Results indicate a peculiar short-chain acylcarnitine profile characterizing buffalo milk, widening the current knowledge about its composition and nutritional value.

Ruminant meat and milk contain δ-valerobetaine, another precursor of trimethylamine N-oxide (TMAO) like γ-butyrobetaine.

Quaternary ammonium compounds containing N-trimethylamino moiety, such as choline derivatives and carnitine, abundant in meat and dairy products, are metabolic precursors of trimethylamine (TMA). A similar fate is reported for Nε-trimethyllysine and γ-butyrobetaine. With the aim at investigating the metabolic profile of such metabolites in most employed animal dietary sources, HPLC-ESI-MS/MS analyses on ruminant and non-ruminant milk and meat were performed. Results demonstrate, for the first time, the presence of δ-valerobetaine, occurring at levels higher than γ-butyrobetaine in all ruminant samples compared to non-ruminants. Demonstration of δ-valerobetaine metabolic origin, surprisingly, showed that it originates from rumen through the transformation of dietary Nε-trimethyllysine. These results highlight our previous findings showing the ubiquity of free Nε-trimethyllysine in vegetable kingdom. Furthermore, δ-valerobetaine, similarly to γ-butyrobetaine, can be degraded by host gut microbiota producing TMA, precursor of the proatherogenic trimethylamine N-oxide (TMAO), unveiling its possible role in the biosynthetic route of TMAO.

Ophthalmic acid is a marker of oxidative stress in plants as in animals.

BACKGROUND: Ophthalmic acid (OPH), γ-glutamyl-L-2-aminobutyryl-glycine, a tripeptide analogue of glutathione (GSH), has recently captured considerable attention as a biomarker of oxidative stress in animals. The OPH and GSH biosynthesis, as well as some biochemical behaviors, are very similar. Here, we sought to investigate the presence of OPH in plants and its possible relationship with GSH, known to possess multiple functions in the plant development, growth and response to environmental changes. METHODS: HPLC-ESI-MS/MS analysis was used to examine the occurrence of OPH in leaves from various plant species, and flours from several plant seeds. Different types of oxidative stress, i.e., water, dark, paraquat, and cadmium stress, were induced in rye, barley, oat, and winter wheat leaves to evaluate the effects on the levels of OPH and its metabolic precursors. RESULTS: OPH and its dipeptide precursor, γ-glutamyl-2-aminobutyric acid, were found to occur in phylogenetically distant plants. Interestingly, the levels of OPH were tightly associated with the oxidative stress tested. Levels of OPH precursors, γ-glutamyl-2-aminobutyric acid and 2-aminobutyric acid, the latter efficiently formed in plants via biosynthetic pathways absent in the animal kingdom, were also found to increase during oxidative stress. CONCLUSIONS: OPH occurs in plants and its levels are tightly associated with oxidative stress. GENERAL SIGNIFICANCE: OPH behaves as an oxidative stress marker and its biogenesis might occur through a biochemical pathway common to many living organisms.

The betaine profile of cereal flours unveils new and uncommon betaines.

We report the LC-ESI-MS/MS determination of betaines in commercial flours of cereals and pseudocereals most utilized in human nutrition. Results showed that glycine betaine, trigonelline, proline betaine, Nε-trimethyllysine were metabolites common to all examined flours, whereas an uncommon betaine, valine betaine, and glutamine betaine were present only in flours of barley, rye, oat, durum wheat, winter wheat, Triticum dicoccum and Triticum monococcum. Valine betaine and glutamine betaine, the latter never reported before in plants and animals, are not evenly distributed in the Poaceae family, but their presence or absence in flours depends on the subfamily to which the plant belongs. Interestingly, we also report for the first time the occurrence of pipecolic acid betaine (homostachydrine) and its precursor 1,2-N-methylpipecolic acid in rye flour. These two metabolites were not detected in any other cereal or pseudocereal flour, suggesting their potential role as markers of rye flour occurrence in cereal-based foods.

1,2,4-Thiadiazolidin-3,5-diones as novel hydrogen sulfide donors.

Hydrogen sulfide (H2S) is an endogenous modulator that plays significant physio-pathological roles in several biological systems. In this research field there is a large interest in developing selective CBS and CSE inhibitors and H2S releasing moieties, that could be either used as therapeutic agents or linked to known drugs. One of the major problem is the limited availability of chemicals that ensure a controlled release of H2S in vitro as well in vivo. Aiming to obtain novel H2S donors, whose release properties could be appropriately modulated, we have synthesized a series of 1,2,4-thiadiazolidine-3,5-diones (THIA 1-10) as innovative donors that could release H2S in controlled manner. All the synthesized compounds were evaluated for their H2S releasing properties by an amperometric approach and for their vasorelaxant ability on aorta rings. In order to rationalize the obtained results, a detailed study on the release mechanism has been performed using the most efficient H2S donor, THIA 3 (Cmax 65.4 µM and EC50 1.7 µM).

Ergothioneine products derived by superoxide oxidation in endothelial cells exposed to high-glucose.

Ergothioneine (Egt), 2-mercapto-L-histidine betaine (ESH), is a dietary component acting as antioxidant and cytoprotectant. In vitro studies demonstrated that Egt, a powerful scavenger of hydroxyl radicals, superoxide anion, hypochlorous acid and peroxynitrite, protects vascular function against oxidative damages, thus preventing endothelial dysfunction. In order to delve the peculiar oxidative behavior of Egt, firstly identified in cell free-systems, experiments were designed to identify the Egt oxidation products when endothelial cells (EC) benefit of its protection against high-glucose (hGluc). HPLC-ESI-MS/MS analyses revealed a decrease in the intracellular GSH levels and an increase in the ophthalmic acid (OPH) levels during hGluc treatment. Interestingly, in the presence of Egt, the decrease of the GSH levels was lower than in cells treated with hGluc alone, and this effect was paralleled by lower OPH levels. Egt was also effective in reducing the cytotoxicity of H2O2 and paraquat (PQT), an inducer of superoxide anion production, showing a similar time-dependent pattern of GSH and OPH levels, although with peaks occurring at different times. Importantly, Egt oxidation generated not only hercynine (EH) but also the sulfonic acid derivative (ESO3H) whose amounts were dependent on the oxidative stress employed. Furthermore, cell-free experiments confirmed the formation of both EH and ESO3H when Egt was reacted with superoxide anion. In summary, these data, by identifying the EH and ESO3H formation in EC exposed to hGluc, highlight the cellular antioxidant properties of Egt, whose peculiar redox behavior makes it an attractive candidate for the prevention of oxidative stress-associated endothelial dysfunction during hyperglycemia.

Tyramine Pathways in Citrus Plant Defense: Glycoconjugates of Tyramine and Its N-Methylated Derivatives.

Glucosylated forms of tyramine and some of its N-methylated derivatives are here reported for the first time to occur in Citrus genus plants. The compounds tyramine-O-ß-d-glucoside, N-methyltyramine-O-ß-d-glucoside, and N,N-dimethyltyramine-O-ß-d-glucoside were detected in juice and leaves of sweet orange, bitter orange, bergamot, citron, lemon, mandarin, and pomelo. The compounds were identified by mass spectrometric analysis, enzymatic synthesis, and comparison with extracts of Stapelia hirsuta L., a plant belonging to the Apocynaceae family in which N,N-dimethyltyramine-O-ß-d-glucoside was identified by others. Interestingly, in Stapelia hirsuta we discovered also tyramine-O-ß-d-glucoside, N-methyltyramine-O-ß-d-glucoside, and the tyramine metabolite, N,N,N-trimethyltyramine-O-ß-glucoside. However, the latter tyramine metabolite, never described before, was not detected in any of the Citrus plants included in this study. The presence of N-methylated tyramine derivatives and their glucosylated forms in Citrus plants, together with octopamine and synephrine, also deriving from tyramine, supports the hypothesis of specific biosynthetic pathways of adrenergic compounds aimed to defend against biotic stress.

Ergothioneine oxidation in the protection against high-glucose induced endothelial senescence: Involvement of SIRT1 and SIRT6.

Ergothioneine (Egt), the betaine of 2-mercapto-L-histidine, is a dietary antioxidant protecting against many diseases, including cardiovascular disease (CVD), through a redox mechanism different from alkylthiols. Here, experiments were designed to evaluate the mechanisms underlying the beneficial effect of Egt against hyperglycaemia-induced senescence in endothelial cells. To this end, cells were incubated with increasing concentrations of Egt (0.01-1.00mM) for 12h followed by incubation for 48h with high-glucose (25mM). Cell evaluation indicated that viability was not affected by mM concentrations of Egt and that the high-glucose cytotoxicity was prevented with the highest efficacy at 0.5mM Egt. The cytoprotective effect of Egt was paralleled by reduced ROS production, cell senescence, and, interestingly, the formation of hercynine (EH), a betaine we recently found to be produced during the Egt oxidation pathway. Notably, the Egt beneficial effect was exerted through the upregulation of sirtuin 1 (SIRT1) and sirtuin 6 (SIRT6) expression and the downregulation of p66Shc and NF-κB. SIRT1 activity inhibition and SIRT6 gene silencing by small interfering RNA abolished the protective effect of Egt against the high-glucose-induced endothelial senescence. These data provide the first evidence of the Egt ability to interfere with endothelial senescence linked to hyperglycaemia through the regulation of SIRT1 and SIRT6 signaling, thus further strengthening the already assessed role of these two histone deacetylases in type 2 diabetes.

Homostachydrine (pipecolic acid betaine) as authentication marker of roasted blends of Coffea arabica and Coffea canephora (Robusta) beans.

The occurrence of pipecolic acid betaine (homostachydrine) and its biosynthetic precursor N-methylpipecolic acid was detected for the first time in green coffee beans of Robusta and Arabica species. The analyses were conducted by HPLC-ESI tandem mass spectrometry and the metabolites identified by product ion spectra and comparison with authentic standards. N-methylpipecolic acid was found at similar levels in green coffee beans of Robusta and Arabica, whereas a noticeable difference of homostachydrine content was observed between the two green coffee bean species. Interestingly, homostachydrine content was found to be unaffected by coffee bean roasting treatment because of a noticeable heat stability, a feature that makes this compound a candidate marker to determine the content of Robusta and Arabica species in roasted coffee blends. To this end, a number of certified pure Arabica and Robusta green beans were analyzed for their homostachydrine content. Results showed that homostachydrine content was 1.5±0.5mg/kg in Arabica beans and 31.0±10.0mg/kg in Robusta beans. Finally, to further support the suitability of homostachydrine as quality marker of roasted blends of Arabica and Robusta coffee beans, commercial samples of roasted ground coffee blends were analyzed and the correspondence between the derived percentages of Arabica and Robusta beans with those declared on packages by manufacturers was verified.

Betaines and related ammonium compounds in chestnut (Castanea sativa Mill.).

Chestnut fruits, being poor of simple sugars and consisting mainly of fibers and starch, are among the constituents of Mediterranean diet. While numerous studies report on content of proteins and amino acids in chestnut, no one has appeared so far on betaines, an important class of nitrogen compounds ubiquitous in plants for their protective action in response to abiotic stress. In this study, we analyzed by HPLC-ESI-tandem mass spectrometry, in fruits and flours of varieties of chestnut cultivated in Italy, the composition of betaines and ammonium compounds intermediates of their biosynthesis. Besides the parent amino acids, the compounds quantified were choline, glycerophosphocholine, phosphocholine, glycine betaine, N-methylproline, proline betaine (stachydrine), ß-alanine betaine, 4-guanidinobutyric acid, trigonelline, N,N,N-trimethyllysine. Interestingly, some uncommon derivatives of pipecolic acid, such as N-methylpipecolic acid, 4-hydroxypipecolic acid and 4-hydroxy-N-methylpipecolic acid were identified for the first time in chestnut samples and characterized by MS(n) tandem mass spectrometry.

Serotonin 5-O-ß-Glucoside and Its N-Methylated Forms in Citrus Genus Plants.

Citrus genus is characterized by a specific presence of indole metabolites deriving from the N-methylation of tryptamine and its hydroxylated form, 5-hydroxytryptamine (serotonin), which are likely involved in plant defense mechanisms. In this study, we identified for the first time the occurrence in Citrus plants of serotonin 5-O-ß-glucoside and all its N-methylated derivatives, that is, N-methylserotonin 5-O-ß-glucoside, N,N-dimethylserotonin (bufotenine) 5-O-ß-glucoside, and N,N,N-trimethylserotonin (bufotenidine) 5-O-ß-glucoside. The identification of the glucosylated compounds was based on mass spectrometric studies, hydrolysis by glucosidase, and in some cases, comparison to authentic compounds. Beside leaves, the distribution of the glucosylated forms and their aglycones in some Citrus species was evaluated in flavedo, albedo, juice, and seeds. The simultaneous presence of serotonin and its N-methylated derivatives, together with the corresponding glucosylated forms, is consistent with the occurrence of a metabolic pathway, specific for Citrus, aimed at potentiating the defensive response to biotic stress through the optimization of the production and use of the most toxic of such metabolites.

Sirtuins in vascular diseases: Emerging roles and therapeutic potential.

Silent information regulator-2 (Sir-2) proteins, or sirtuins, are a highly conserved protein family of histone deacetylases that promote longevity by mediating many of the beneficial effects of calorie restriction which extends life span and reduces the incidence of cancer, cardiovascular disease (CVD), and diabetes. Here, we review the role of sirtuins (SIRT1-7) in vascular homeostasis and diseases by providing an update on the latest knowledge about their roles in endothelial damage and vascular repair mechanisms. Among all sirtuins, in the light of the numerous functions reported on SIRT1 in the vascular system, herein we discuss its roles not only in the control of endothelial cells (EC) functionality but also in other cell types beyond EC, including endothelial progenitor cells (EPC), smooth muscle cells (SMC), and immune cells. Furthermore, we also provide an update on the growing field of compounds under clinical evaluation for the modulation of SIRT1 which, at the state of the art, represents the most promising target for the development of novel drugs against CVD, especially when concomitant with type 2 diabetes.

An uncommon redox behavior sheds light on the cellular antioxidant properties of ergothioneine.

Ergothioneine (ESH), an aromatic thiol occurring in the human diet and which accumulates in particular cells, is believed to act as an antioxidant. However, its redox mechanism remains unclear and it does not seem to provide any advantage compared to other antioxidants, such as alkylthiols, which are better reducing agents and generally present in cells at higher levels. Here, we investigated by ESI-MS the products of ESH oxidation produced by neutrophils during oxidative burst and, to further elucidate ESH redox behavior, we also analyzed the oxidation products of the reaction of ESH with hypochlorite in cell-free solutions. Indeed, neutrophils are the main source of hypochlorite in humans. Furthermore, we also tested other biologically relevant oxidants, such as peroxynitrite and hydrogen peroxide. Our results indicate that treatment of human neutrophils with phorbol 12-myristate 13-acetate in the presence of ESH leads to a remarkable production of the sulfonated form (ESO3H), a compound never described before, and hercynine (EH), the desulfurated form of ESH. Similar results were obtained when ESH was subjected to cell-free oxidation in the presence of hypochlorite, as well as hydrogen peroxide or peroxynitrite. Furthermore, when the disulfide of ESH was reacted with those oxidants, we found that it was also oxidized, with production of EH and ESO3H, whose amount was dependent on the oxidant strength. These data reveal a unique ESH redox behavior, entirely different from that of alkylthiols, and suggest a mechanism, so far overlooked, through which ESH performs its antioxidant action in cells.

N-methylated derivatives of tyramine in citrus genus plants: identification of N,N,N-trimethyltyramine (candicine).

The distribution of tyramine and its methylated derivatives, N-methyltyramine and N,N-dimethyltyramine, was investigated in tissue parts (leaves and fruits) of several plants of Citrus genus by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In the course of our study we discovered the occurrence of N,N,N-trimethyltyramine in all citrus plants examined. This quaternary ammonium compound, known to act in animals as a neurotoxin, was recognized and characterized by mass spectrometric analysis. The substance, never described before in the Citrus genus, is also known as candicine or maltoxin. Results indicate that N,N,N-trimethyltyramine is consistently expressed in leaves of clementine, bitter orange, and lemon. Conversely, low levels were found in the leaves of orange, mandarin, chinotto (Citrus myrtifolia), bergamot, citron, and pomelo. In the edible part of the fruits, N,N,N-trimethyltyramine was found at trace levels.

Determination of homoarginine, arginine, NMMA, ADMA, and SDMA in biological samples by HPLC-ESI-mass spectrometry.

N(G),N(G)-dimethyl-L-arginine (ADMA) and N(G)-methyl-L-arginine (NMMA) are endogenous inhibitors of nitric oxide synthase (NOS). In contrast, N(G),N'(G)-dimethyl-L-arginine (SDMA) possesses only a weak inhibitory potency towards neuronal NOS and it is known to limit nitric oxide (NO) production by competing with L-arginine for cellular uptake. The inhibition of NOS is associated with endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. L-homoarginine (HArg), a structural analog of L-arginine (Arg), is an alternative but less efficient substrate for NOS. Besides, it inhibits arginase, leading to an increased availability of L-arginine for NOS to produce NO. However, its relation with cardiovascular disease remains unclear. To date, several analytical methods for the quantitative determination of Arg, HArg, NMMA, AMDA, and SDMA in biological samples have been described. Here, we present a simple, fast, and accurate HPLC-ESI-MS/MS method which allows both the simultaneous determination and quantification of these compounds without needing derivatization, and the possibility to easily modulate the chromatographic separation between HArg and NMMA (or between SDMA and ADMA). Data on biological samples revealed the feasibility of the method, the minimal sample preparation, and the fast run time which make this method very suitable and accurate for analysis in the basic and clinical settings.

Citrus genus plants contain N-methylated tryptamine derivatives and their 5-hydroxylated forms.

The occurrence and distribution in Citrus genus plants of N-methylated derivatives of tryptamine and their 5-hydroxylated forms are reported. Tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, N,N,N-trimethyltryptamine, 5-hydroxytryptamine (serotonin), 5-hydroxy-N-methyltryptamine, 5-hydroxy-N,N-dimethyltryptamine (bufotenine), and 5-hydroxy-N,N,N-trimethyltryptamine (bufotenidine) were quantitated by LC-ESI-MS/MS. Leaves of all citrus plants examined contained N,N,N-trimethyltryptamine, a compound that we first discovered in the bergamot plant. Interestingly, we also found out that all plants examined contained 5-hydroxy-N,N-dimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine, compounds never described so far in the Citrus genus. As N,N,N-trimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine possess nicotine-like activity by exerting their action on acetylcholine receptors, it is conceivable that both represent the arrival point of a biosynthetic pathway aimed to provide Citrus plants with chemical defense against aggressors. This hypothesis is supported by our finding that leaves and seeds, which are more frequently attacked by biotic agents, are the parts of the plant where the highest levels of those compounds were found.

Biodegradability, toxicity and mutagenicity of detergents: Integrated experimental evaluations.

The widespread use of detergents has raised concern with regard to the environmental pollution caused by their active ingredients, which are biorefractory, toxic and persistent. Since detergents are complex mixtures of different substances, in which synergistic effects may occur, we aimed to assess the mutagenicity of different detergent formulations, taking into account aquatic toxicity and ready biodegradability. We performed a ready biodegradability test (OECD 301 F), Daphnia magna and Vibrio fischeri toxicity tests, and mutagenicity tests (Salmonella/microsome test, Allium cepa test and comet assay). Six detergent formulations were examined, 3 pre-manufacture and 3 commercially available. All detergents presented ready biodegradability. EC50 values varied for all products, according to the marker organism used, but were always higher than the more stringent value considered for aquatic toxicity assessment (V. fischeri 10-60 mg/L; D. magna 25-300 mg/L; A. cepa 250-2000 mg/L). None of the detergents caused mutations in bacteria. However, one commercial ecolabelled product induced an increase in micronucleus frequency in A. cepa root cells. All pre-manufacture detergents and one commercial one, which gave negative results in the Ames and A. cepa tests, induced DNA damage in human leukocytes. A more accurate evaluation of the environmental impact of complex mixtures such as detergents requires a battery of tests to describe degradation, as well as toxicological and mutagenic features.