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Sample manipulation for storage and storage itself, interfere with the stability of labile lipids in human plasma, including vitamin E (α-tocopherol), polyunsaturated fatty acids (PUFAs), and their enzymatic and free radical-derived oxidation metabolites. This remains a main limit of lipidomics studies that often lack of sufficient standardization and validation at the pre-analytical level. In order to characterize the stability of these lipids in human plasma and to develop a standardized pre-analytical protocol for lipidomics methods, the oxidation metabolites of α-tocopherol, the free form of ω3 and ω6 PUFAs, and some arachidonic acid (AA)-derived eicosanoids were investigated in human plasma during storage at different freezing temperatures. The effect of a protection/defense cocktail of antioxidants and lipoxygenase inhibitors (PD solution) on these lipid parameters was also evaluated. The temperature of storage markedly affected the formation of α-tocopheryl quinone (α-TQ), the main lipoperoxyl radical-derived oxidation metabolite of vitamin E, with the lowest production rate observed in samples stored at −80 °C or in liquid nitrogen. A similar effect of the storage temperature was observed for the free form of the ω-3 species eicosapentaenoic and docosahexaenoic acid, and for the ω-6 AA. Freezing samples at −20 °C resulted in a time-dependent formation of the pro-inflammatory eicosanoid LTB4. The PD solution prevents non-specific alterations of these lipid parameters in samples that are processed for direct analysis and protects from the temperature-dependent modifications of free PUFAs. Combining PD solution and preservation at −80 °C or in liquid nitrogen, resulted in levels of α-TQ and PUFAs that remained stable over 1 month and up to 8 months of storage, respectively. This method paper provides indications for the optimal processing and storage of human plasma utilized in lipidomics studies.

The metabolic syndrome (MetSyn) is a disorder characterized by a cluster of diseases where the regulation of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor endowed with ligand- and context-dependent activities, can have a major therapeutic relevance. We have recently discovered a tryptophan metabolite of microbial origin, indole-3-carboxaldehyde (3-IAld), able to regulate intestinal mucosal homeostasis by acting as a ligand of AhR. This makes 3-IAld a potential candidate to treat MetSyn related ailments. Herein, we provide a proof of concept on the efficacy and safety of 3-IAld encapsulated in enteric microparticles (MP) in vivo in a MetSyn murine model. In particular, we showed that 3-IAld: i) is released from MPs in the intestine and potentially metabolized; ii) is able to activate AhR locally; iii) prevents the metabolic complications and the intestinal epithelial barrier dysfunction; iv) is not associated with toxic events. This study does not only extend the biological activity of 3-IAld in vivo, but also provides hints on the therapeutic potential of 3-IAld delivered by enteric MP in MetSyn related diseases.

The metabolism of &alpha, tocopherol (&alpha, 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 &alpha, TOH so far identified in human blood, i.e., the &ldquo, vitamin E metabolome&rdquo, 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 &alpha, TOH intake. To this end, a one-week oral administration protocol with 800 U RRR-&alpha, TOH/day was performed in 17 healthy volunteers, and &alpha, 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 &alpha, 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 &alpha, CEHC (carboxy-ethyl-hydroxychroman) and the long-chain metabolites M1 and &alpha, 13&prime, OH, such variability was found to interfere with the possibility to utilize them as sensitive indicators of &alpha, TOH intake. On the contrary, the free radical-derived metabolite &alpha, tocopheryl quinone significantly correlated with the post-supplementation levels of &alpha, TOH. The supplementation stimulated PXR, but not CYP4F2, expression of leucocytes, and significant correlations were observed between the baseline levels of &alpha, TOH and both the baseline and post-supplementation levels of PXR. These findings provide original analytical and molecular information regarding the human metabolism of &alpha, TOH and its intrinsic variability, which is worth considering in future nutrigenomics and interventions studies.

A multiclass method for the determination of antimicrobial substances in eggs has been developed and validated, covering sixty-three antibiotics belonging to ten families. After extraction with acidified acetonitrile and EDTA, the analytes were injected into a liquid-chromatography high-resolution mass-spectrometry system operating in positive ionization mode. The procedure was successfully validated as confirmatory method evaluating selectivity, linearity, precision, recovery, decision limit, detection capability, limits of detection and quantitation. The here developed is the first method including all the antibiotics with Maximum Residue Limit (MRL) in eggs, except very polar drugs (aminoglycosides and colistins), which require specific protocols. Finally, a wide survey was carried out analyzing 100 commercial eggs samples from local markets. Only 3% contained residues of authorized substances at concentrations lower than the relevant MRLs.

This study aims to investigate in in vivo and in vitro models of nonalcoholic fatty liver disease (NAFLD) the enzymatic metabolism of α-tocopherol (vitamin E) and its relationship to vitamin E-responsive genes with key role in the lipid metabolism and detoxification of the liver. The experimental models included mice fed a high-fat diet combined or not with fructose (HFD+F) and HepG2 human hepatocarcinoma cells treated with the lipogenic agents palmitate, oleate or fructose. CYP4F2 protein, a cytochrome P-450 isoform with proposed α-tocopherol ω-hydroxylase activity, decreased in HFD and even more in HFD+F mice liver; this finding was associated with increased hepatic levels of α-tocopherol and decreased formation of the corresponding long-chain metabolites α-13-hydroxy and α-13-carboxy chromanols. A decreased expression was also observed for PPAR-γ and SREBP-1 proteins, two vitamin E-responsive genes with key role in lipid metabolism and CYP4F2 gene regulation. A transient activation of CYP4F2 gene followed by a repression response was observed in HepG2 cells during the exposure to increasing levels of the lipogenic and cytotoxic agent palmitic acid; such gene repression effect was further exacerbated by the co-treatment with oleic acid and α-tocopherol and was also observed for PPAR-γ and the SREBP isoforms 1 and 2. Such gene response was associated with increased uptake and ω-hydroxylation of α-tocopherol, which suggests a minor role of CYP4F2 in the enzymatic metabolism of vitamin E in HepG2 cells. In conclusion, the liver metabolism and gene response of α-tocopherol are impaired in experimental NAFLD.

The role of vitamin E in both enzymatic and free radical-dependent metabolism of polyunsaturated fatty acids (PUFAs) has been well demonstrated. This study proposed a new LC-MS/MS method to quantify the main vitamin E forms, their metabolites and main PUFA species in human blood, since, at present, there are not procedures able to simultaneously determine these two classes of compounds. After the optimization of sample treatment and reverse-phase separation conditions, tandem mass spectrometry detection was evaluated experimenting both positive and negative electrospray ionisation modes. The procedure was also preliminarily adapted to assess five arachidonic acid-derived eicosanoids that could be under the influence of vitamin E function, such as LTB4 (leukotriene B4), 20-HETE (20-hydroxyeicosatetraenoic acid) and their ω-oxidation metabolites. After the validation study, the performance characteristics were confirmed analysing a certified reference material (SRM® 1950 - frozen human plasma by NIST). Finally, an application of the method in the analysis of lipid abnormalities of chronic kidney disease patients was shown.

The expression of CYP4F2, a form of cytochrome P-450 with proposed role in α-tocopherol and long-chain fatty acid metabolism, was explored in HepG2 and HepaRG human hepatocytes during ethanol toxicity. Cytotoxicity, ROS production, and JNK and ERK1/2 kinase signaling increased in a dose and time-dependent manner during ethanol treatments; CYP4F2 gene expression decreased, while other CYP4F forms, namely 4F11 and 12, increased along with 3A4 and 2E1 isoforms. α-Tocopherol antagonized the cytotoxicity and CYP4F2 gene repression effect of ethanol in HepG2 cells. Ethanol stimulated the tocopherol-ω-hydroxylase activity and the other steps of vitamin E metabolism, which points to a minor role of CYP4F2 in this metabolism of human hepatocytes. PPAR-γ and SREBP-1c followed the same expression pattern of CYP4F2 in response to ethanol and α-tocopherol treatments. Moreover, the pharmacological inhibition of PPAR-γ synergized with ethanol in decreasing CYP4F2 protein expression, which suggests a role of this nuclear receptor in CYP4F2 transcriptional regulation. In conclusion, ethanol toxicity modifies the CYP expression pattern of human hepatic cells impairing CYP4F2 transcription and protein expression. These changes were associated with a lowered expression of the fatty acid biosynthesis regulators PPAR-γ and SREBP-1c, and with an increased enzymatic catabolism of vitamin E. CYP4F2 gene repression and a sustained vitamin E metabolism appear to be independent effects of ethanol toxicity in human hepatocytes.