In-depth characterization of phenolic profiling of Moraiolo extra-virgin olive oil extract and initial investigation of the inhibitory effect on Indoleamine-2,3-Dioxygenase (IDO1) enzyme.

In this research, the phenolic extract from Moraiolo extra-virgin olive oil (EVOO) was thoroughly characterized. A reversed-phase HPLC method with a photodiode detector allowed to measure a total phenol content higher than 500 mg/kg EVOO, with elevated amounts of oleocanthal, oleacein, and oleouropein aglycone (131.2, 213.5, and 158.4 mg/kg EVOO, respectively). Appreciable amounts of (+)-pinoresinol and (+)- 1-acetoxy-pinoresinol, 3.2 and 12.5 mg/kg EVOO respectively, were measured. High-resolution mass spectrometry with Orbitrap mass analyzer technology was used to confirm the identity of the analytes. Afterwards, the extract was tested, for the first time, for its activity on Indoleamine-2,3-Dioxygenase (IDO1). This enzyme appears as a promising target for the modulation of the neuroinflammatory-oxidative processes relying on the pathogenesis of several neurodegenerative diseases. The extract showed an inhibitory effect on the catalytic activity of both human and murine IDO1, with a good safety at the concentrations of 15 and 30 µg/mL.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs): An optimized LC-MS/MS procedure for feed analysis.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are extremely stable highly fluorinated aliphatic compounds, widely used in chemical and industrial applications since 1950s. They are ubiquitously detected in the environment and diet is the main route for human exposure. To ensure human safety, it is necessary to follow the whole food production chain, including animal feed. Still PFASs are not regulated as undesirable substances in feed, although several studies have shown they can be transferred from feed to livestock and thereafter to food. This paper describes the development, optimization and the full validation of a sensitive and reliable analytical protocol enabling the quantification of 19 PFASs in animal feeds by liquid chromatography - mass spectrometry (LC-MS/MS). The method was optimized, assessing the possible matrix interferences, and submitted to comprehensive validation (55 independent spiking experiments). Validation experiments were conducted on blank fish feed samples (natural levels of PFASs < 0.10 ng g-1) spiked at five different concentrations (0.10, 0.50, 1.0, 5.0 and 10 ng g-1). Apparent recoveries (R%) were generally between 88 and 111%; R% < 80% were obtained only at the lower validation levels for those molecules not having the corresponding labelled analogues. Relative standard deviations in repeatability conditions (RSDr) and within-lab reproducibility conditions (RSDwR) were lower or equal to 11% and 22% respectively. Limits of quantification were set for most of the analytes at 0.10 ng g-1 (LOQs) and verified with repeated analysis on fortified samples (0.10 ng g-1). Limits of detection (LODs) were calculated as 1/3 LOQ. Finally the method was applied to 23 feed samples of different type (compound feed, vegetable feed material, complementary feed, pre-mixture and mineral feed) and all of them did not show PFAS above LOQ.

Alpha-Tocopherol Metabolites (the Vitamin E Metabolome) and Their Interindividual Variability during Supplementation.

The metabolism of α-tocopherol (α-TOH, vitamin E) shows marked interindividual variability, which may influence the response to nutritional and therapeutic interventions with this vitamin. Recently, new metabolomics protocols have fostered the possibility to explore such variability for the different metabolites of α-TOH so far identified in human blood, i.e., the "vitamin E metabolome", some of which have been reported to promote important biological functions. Such advances prompt the definition of reference values and degree of interindividual variability for these metabolites at different levels of α-TOH intake. To this end, a one-week oral administration protocol with 800 U RRR-α-TOH/day was performed in 17 healthy volunteers, and α-TOH metabolites were measured in plasma before and at the end of the intervention utilizing a recently validated LC-MS/MS procedure; the expression of two target genes of α-TOH with possible a role in the metabolism and function of this vitamin, namely pregnane X receptor (PXR) and the isoform 4F2 of cytochrome P450 (CYP4F2) was assessed by immunoblot in peripheral blood leukocytes. The levels of enzymatic metabolites showed marked interindividual variability that characteristically increased upon supplementation. With the exception of α-CEHC (carboxy-ethyl-hydroxychroman) and the long-chain metabolites M1 and α-13'OH, such variability was found to interfere with the possibility to utilize them as sensitive indicators of α-TOH intake. On the contrary, the free radical-derived metabolite α-tocopheryl quinone significantly correlated with the post-supplementation levels of α-TOH. The supplementation stimulated PXR, but not CYP4F2, expression of leucocytes, and significant correlations were observed between the baseline levels of α-TOH and both the baseline and post-supplementation levels of PXR. These findings provide original analytical and molecular information regarding the human metabolism of α-TOH and its intrinsic variability, which is worth considering in future nutrigenomics and interventions studies.

Optimized one-pot derivatization and enantioseparation of cysteine: Application to the study of a dietary supplement.

Cysteine is a sulfur-containing amino acid which plays an outstanding role in many biological pathways in mammals. The analysis and quantification of native cysteine remains a critical issue due to its highly reactive thiol group evolving to the disulfide cystine derivative through oxidation reaction. Aimed at improving the derivative stability, cysteine was labelled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), which reacts with both amino and thiol groups. The derivatization was optimized and the chemical identity of the reaction product was assessed via high-resolution mass spectrometry. The NBD-cysteine derivative resulted stable for 10 days. This derivative was enantioresolved (α and RS equal to 1.25 and 2.70, respectively) thanks to a (R,R)-Whelk-O1 phase with the following chromatographic setting: eluent, MeOH/water-90/10 (v/v) with 15 mM ammonium formate (pwsH 6.0); column temperature, 35 °C; flow rate, 1.0 mL/min. The developed method was validated following the ICH guidelines and applied for the quality control of a L-cysteine containing dietary supplement.

Nutritional and lipidomics biomarkers of docosahexaenoic acid-based multivitamin therapy in pediatric NASH.

Two recent randomized controlled trials demonstrated improved radiographic, histological and hepatometabolic cues of non-alcoholic steatohepatitis (NASH) in pediatric patients treated with the ω-3 fatty acid docosahexaenoic acid (DHA) in combination with vitamin D (VD) or with choline (CHO) and vitamin E (VE), the DHA-VD and DHA-CHO-VE trials, respectively). In the present study we verified the nutritional compliance to these DHA-based multivitamin treatments; lipidomics biomarkers of the reported outcome on NASH indicators were also investigated. Samples were obtained from 30 biopsy-proven pediatric NASH patients of the DHA-CHO-VE trial randomized in multivitamin treatment group and placebo group (n = 15 each), and from 12 patients of the treatment group of the DHA-VD trial. All patients underwent 6-month therapy plus 6 months of follow-up. Plasma samples and clinical data were obtained at baseline and at the end of the study (12 months). Selected biomarkers included the free form of DHA and other ω-3 fatty acid arachidonic acid (AA), indices of the vitamin E status, and some hepatic metabolites of these lipids. Radiographic and histological improvements of treated patients were associated with increased concentrations of DHA, α-linolenic acid and α-tocopherol (i.e. VE), and with decreased AA that was also investigated in complex lipids by untargetd lipidomics. As a result a significantly lowered AA/DHA ratio was observed to represent the main indicator of the response to the DHA-based therapy. Furthermore, baseline levels of AA/DHA showed strong association with NAS and US improvement. A stable correction of DHA AA metabolism interaction is associated with the curative effect of this therapy and may represent a key nutritional endpoint in the clinical management of pediatric NASH.

Increased plasma levels of the lipoperoxyl radical-derived vitamin E metabolite α-tocopheryl quinone are an early indicator of lipotoxicity in fatty liver subjects.

Lipid peroxidation is one of the earliest pathogenic events of non-alcoholic fatty liver disease (NAFLD). In this context, an increased oxidation of the lipoperoxyl radical scavenger α-tocopherol (α-TOH) should occur already in the subclinical phases of the disease to compensate for the increase oxidation of the lipid excess of liver and possibly of other tissues. However, this assumption remains unsupported by direct analytical evidence. In this study, GC-MS/MS and LC-MS/MS procedures have been developed and applied for the first time to measure the vitamin E oxidation metabolite α-tocopheryl quinone (α-TQ) in plasma of fatty liver (FL) subjects that were compared in a pilot cross-sectional study with healthy controls. The protein adducts of 4-hydroxynonenal (4-HNE) and the free form of polyunsaturated free fatty acids (PUFA) were measured as surrogate indicators of lipid peroxidation. α-TQ formation was also investigated in human liver cells after supplementation with α-TOH and/or fatty acids (to induce steatosis). Compared with controls, FL subjects showed increased (absolute and α-TOH-corrected) levels of plasma α-TQ and 4-HNE, and decreased concentrations of PUFA. α-TQ levels positively correlated with indices of liver damage and metabolic dysfunction, such as alanine aminotransferase, bilirubin and triglycerides, and negatively correlated with HDL cholesterol. Fatty acid supplementation in human hepatocytes stimulated the generation of cellular oxidants and α-TOH uptake leading to increased α-TQ formation and secretion in the extracellular medium - both were markedly stimulated by α-TOH supplementation. In conclusion, plasma α-TQ represents an early biomarker of the lipoperoxyl radical-induced oxidation of vitamin E and lipotoxicity of the fatty liver.

Determination of tocopherols and their metabolites by liquid-chromatography coupled with tandem mass spectrometry in human plasma and serum.

Several studies are increasingly underlying the biological role of vitamin E metabolites as bioactive compounds with anti-inflammatory, anti-proliferative and anti-atherogenic activity. A quantitative method for the simultaneous determination in human plasma and serum of vitamin E (α-tocopherol, α-T and γ-tocopherol, γ-T) and its cytochrome P-450 metabolites: 13'-hydroxychromanol (α-13'-OH), 13'-carboxychromanol (α-13'-COOH) and carboxyethyl hydroxychromanols (α-CEHC and γ-CEHC), was developed and validated. After enzymatic hydrolysis and deproteinization, the metabolites were extracted with a mixture of hexane/ methyl tertiary butyl ether (2/1, v/v). The separation was achieved by reversed phase chromatography and the analytes detected by a triple quadrupole mass analyser using electrospray ionization in positive mode (LC-MS/MS). α-T and γ-T were extracted separately without enzymatic hydrolysis. The analytes were quantified with the isotopic dilution method. After an extensive validation study (three levels in three different occasions for a total of 54 experiments), the procedure was successfully applied to the analysis of sera of healthy volunteers (before and after supplementation with α-T) and plasma of patients affected by chronic kidney disease. Finally, the structures of three unknown compounds found in blood and related to the long chain metabolites (α-13'-OH and α-13'-COOH) were further investigated using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS).