Convenient and Sensitive Measurement of Lactosylceramide Synthase Activity Using Deuterated Glucosylceramide and Mass Spectrometry

Lactosylceramide is necessary for the biosynthesis of almost all classes of glycosphingolipids and plays a relevant role in pathways involved in neuroinflammation. It is synthesized by the action of galactosyltransferases B4GALT5 and B4GALT6, which transfer galactose from UDP-galactose to glucosylceramide. Lactosylceramide synthase activity was classically determined in vitro by a method based on the incorporation of radiolabeled galactose followed by the chromatographic separation and quantitation of the product by liquid scintillation counting. Here, we used deuterated glucosylceramide as the acceptor substrate and quantitated the deuterated lactosylceramide product by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We compared this method with the classical radiochemical method and found that the reactions have similar requirements and provide comparable results in the presence of high synthase activity. Conversely, when the biological source lacked lactosylceramide synthase activity, as in the case of a crude homogenate of human dermal fibroblasts, the radiochemical method failed, while the other provided a reliable measurement. In addition to being very accurate and sensitive, the proposed use of deuterated glucosylceramide and LC-MS/MS for the detection of lactosylceramide synthase in vitro has the relevant advantage of avoiding the costs and discomforts of managing radiochemicals

Impact of lipid sources on quality traits of medical cannabis-based oil preparations

The feasibility of the use of two lipid sources and their impact on the cannabinoid profile, terpene fingerprint, and degradation products in medical cannabis oil preparations during 3 months of refrigerated storage time were investigated. LCHRMS-Orbitrap® and HS-SPME coupled to GC-MS for the investigation of targeted and untargeted cannabinoids, terpenes, and lipid degradation products in Bedrocan® and Bediol® macerated oils were used as analytical approaches. As regards the cannabinoid trend during 90 days of storage, there were no differences between PhEur-grade olive oil (OOPH) and medium-chain triglycerides oil (MCT oil) coupled to a good stability of preparations for the first 60 days both in Bedrocan® and Bediol® oils. MCT lipid source extracted a significant concentration of terpenes compared to olive oil. Terpenes showed a different scenario since MCT oil displayed the strongest extraction capacity and conservation trend of all compounds during the shelf life. Terpenes remained stable throughout the entire storage period in MCT formulations while a significant decrease after 15 and 30 days in Bediol® and Bedrocan® was observed in olive oil. Therefore, MCT oil could be considered a more suitable lipid source compared to olive oil involved in the extraction of medical cannabis for magistral preparations.Fil: Ramella, Alberto. Farmacia Dott.ri Giuliana E Alberto Ramellasas; ItaliaFil: Roda, Gabriella. Università degli Studi di Milano; ItaliaFil: Pavlovic, Radmila. Universita Degli Studi Di Milano. Medicina Veterinaria. Dipartimento Di Scienze Veterinarie Per la Salute.; ItaliaFil: Cas, Michele Dei. Università degli Studi di Milano; ItaliaFil: Casagni, Eleonora. Università degli Studi di Milano; ItaliaFil: Mosconi, Giacomo. Universita Degli Studi Di Milano. Medicina Veterinaria. Dipartimento Di Scienze Veterinarie Per la Salute.; ItaliaFil: Cecati, Francisco Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Minghetti, Paola. Università degli Studi di Milano; ItaliaFil: Grizzetti, Carlo. Ospedale Di Circolofondazione Macchi, Asst Sette Laghi; Itali

Myriocin modulates the altered lipid metabolism and storage in cystic fibrosis.

Cystic fibrosis (CF) is a hereditary disease mostly related to ΔF508 CFTR mutation causing a proteinopathy that is characterized by multiple organ dysfunction, primarily lungs chronic inflammation, and infection. Defective autophagy and accumulation of the inflammatory lipid ceramide have been proposed as therapeutic targets. Accumulation of lipids and cholesterol was reported in the airways of CF patients, together with altered triglycerides and cholesterol levels in plasma, thus suggesting a disease-related dyslipidemia. Myriocin, an inhibitor of sphingolipids synthesis, significantly reduces inflammation and activates TFEB-induced response to stress, enhancing fatty acids oxidation and promoting autophagy. Myriocin ameliorates the response against microbial infection in CF models and patients' monocytes. Here we show that CF broncho-epithelial cells exhibit an altered distribution of intracellular lipids. We demonstrated that lipid accumulation is supported by an enhanced synthesis of fatty acids containing molecules and that Myriocin is able to reduce such accumulation. Moreover, Myriocin modulated the transcriptional profile of CF cells in order to restore autophagy, activate an anti-oxidative response, stimulate lipid metabolism and reduce lipid peroxidation. Moreover, lipid storage may be altered in CF cells, since we observed a reduced expression of lipid droplets related proteins named perilipin 3 and 5 and seipin. To note, Myriocin up-regulates the expression of genes that are involved in lipid droplets biosynthesis and maturation. We suggest that targeting sphingolipids de novo synthesis may counteract lipids accumulation by modulating CF altered transcriptional profile, thus restoring autophagy and lipid metabolism homeostasis

Erratum: Inflammatory extracellular vesicles prompt heart dysfunction via TLR4-dependent NF-κB activation: Erratum.

[This corrects the article DOI: 10.7150/thno.39072.]

Increased Sensitivity of Computed Tomography Scan for Neoplastic Tissues Using the Extracellular Vesicle Formulation of the Contrast Agent Iohexol.

Computed tomography (CT) is a diagnostic medical imaging modality commonly used to detect disease and injury. Contrast agents containing iodine, such as iohexol, are frequently used in CT examinations to more clearly differentiate anatomic structures and to detect and characterize abnormalities, including tumors. However, these contrast agents do not have a specific tropism for cancer cells, so the ability to detect tumors is severely limited by the degree of vascularization of the tumor itself. Identifying delivery systems allowing enrichment of contrast agents at the tumor site would increase the sensitivity of detection of tumors and metastases, potentially in organs that are normally inaccessible to contrast agents, such as the CNS. Recent work from our laboratory has identified cancer patient-derived extracellular vesicles (PDEVs) as effective delivery vehicles for targeting diagnostic drugs to patients' tumors. Based on this premise, we explored the possibility of introducing iohexol into PDEVs for targeted delivery to neoplastic tissue. Here, we provide preclinical proof-of-principle for the tumor-targeting ability of iohexol-loaded PDEVs, which resulted in an impressive accumulation of the contrast agent selectively into the neoplastic tissue, significantly improving the ability of the contrast agent to delineate tumor boundaries

Effect of Coffee and Cocoa-Based Confectionery Containing Coffee on Markers of DNA Damage and Lipid Peroxidation Products: Results from a Human Intervention Study

The effect of coffee and cocoa on oxidative damage to macromolecules has been investigated in several studies, often with controversial results. This study aimed to investigate the effect of one-month consumption of different doses of coffee or cocoa-based products containing coffee on markers of DNA damage and lipid peroxidation in young healthy volunteers. Twenty-one volunteers were randomly assigned into a three-arm, crossover, randomized trial. Subjects were assigned to consume one of the three following treatments: one cup of espresso coffee/day (1C), three cups of espresso coffee/day (3C), and one cup of espresso coffee plus two cocoa-based products containing coffee (PC) twice per day for 1 month. At the end of each treatment, blood samples were collected for the analysis of endogenous and H(2)O(2)-induced DNA damage and DNA oxidation catabolites, while urines were used for the analysis of oxylipins. On the whole, four DNA catabolites (cyclic guanosine monophosphate (cGMP), 8-OH-2′-deoxy-guanosine, 8-OH-guanine, and 8-NO2-cGMP) were detected in plasma samples following the one-month intervention. No significant modulation of DNA and lipid damage markers was documented among groups, apart from an effect of time for DNA strand breaks and some markers of lipid peroxidation. In conclusion, the consumption of coffee and cocoa-based confectionery containing coffee was apparently not able to affect oxidative stress markers. More studies are encouraged to better explain the findings obtained and to understand the impact of different dosages of these products on specific target groups

Effect of Coffee and Cocoa-Based Confectionery Containing Coffee on Markers of DNA Damage and Lipid Peroxidation Products: Results from a Human Intervention Study

The effect of coffee and cocoa on oxidative damage to macromolecules has been investigated in several studies, often with controversial results. This study aimed to investigate the effect of one-month consumption of different doses of coffee or cocoa-based products containing coffee on markers of DNA damage and lipid peroxidation in young healthy volunteers. Twenty-one volunteers were randomly assigned into a three-arm, crossover, randomized trial. Subjects were assigned to consume one of the three following treatments: one cup of espresso coffee/day (1C), three cups of espresso coffee/day (3C), and one cup of espresso coffee plus two cocoa-based products containing coffee (PC) twice per day for 1 month. At the end of each treatment, blood samples were collected for the analysis of endogenous and H2O2-induced DNA damage and DNA oxidation catabolites, while urines were used for the analysis of oxylipins. On the whole, four DNA catabolites (cyclic guanosine monophosphate (cGMP), 8-OH-2′-deoxy-guanosine, 8-OH-guanine, and 8-NO2-cGMP) were detected in plasma samples following the one-month intervention. No significant modulation of DNA and lipid damage markers was documented among groups, apart from an effect of time for DNA strand breaks and some markers of lipid peroxidation. In conclusion, the consumption of coffee and cocoa-based confectionery containing coffee was apparently not able to affect oxidative stress markers. More studies are encouraged to better explain the findings obtained and to understand the impact of different dosages of these products on specific target groups

Convenient and Sensitive Measurement of Lactosylceramide Synthase Activity Using Deuterated Glucosylceramide and Mass Spectrometry

Lactosylceramide is necessary for the biosynthesis of almost all classes of glycosphingolipids and plays a relevant role in pathways involved in neuroinflammation. It is synthesized by the action of galactosyltransferases B4GALT5 and B4GALT6, which transfer galactose from UDP-galactose to glucosylceramide. Lactosylceramide synthase activity was classically determined in vitro by a method based on the incorporation of radiolabeled galactose followed by the chromatographic separation and quantitation of the product by liquid scintillation counting. Here, we used deuterated glucosylceramide as the acceptor substrate and quantitated the deuterated lactosylceramide product by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We compared this method with the classical radiochemical method and found that the reactions have similar requirements and provide comparable results in the presence of high synthase activity. Conversely, when the biological source lacked lactosylceramide synthase activity, as in the case of a crude homogenate of human dermal fibroblasts, the radiochemical method failed, while the other provided a reliable measurement. In addition to being very accurate and sensitive, the proposed use of deuterated glucosylceramide and LC-MS/MS for the detection of lactosylceramide synthase in vitro has the relevant advantage of avoiding the costs and discomforts of managing radiochemicals

An Update on Sphingolipidomics: Is Something Still Missing? Some Considerations on the Analysis of Complex Sphingolipids and Free-Sphingoid Bases in Plasma and Red Blood Cells

The main concerns in targeted “sphingolipidomics” are the extraction and proper handling of biological samples to avoid interferences and achieve a quantitative yield well representing all the sphingolipids in the matrix. Our work aimed to compare different pre-analytical procedures and to evaluate a derivatization step for sphingoid bases quantification, to avoid interferences and improve sensitivity. We tested four protocols for the extraction of sphingolipids from human plasma, at different temperatures and durations, and two derivatization procedures for the conversion of sphingoid bases into phenylthiourea derivatives. Different columns and LC-MS/MS chromatographic conditions were also tested. The protocol that worked better for sphingolipids analysis involved a single-phase extraction in methanol/chloroform mixture (2:1, v/v) for 1 h at 38 °C, followed by a 2 h alkaline methanolysis at 38 °C, for the suppression of phospholipids signals. The derivatization of sphingoid bases promotes the sensibility of non-phosphorylated species but we proved that it is not superior to a careful choice of the appropriate column and a full-length elution gradient. Our procedure was eventually validated by analyzing plasma and erythrocyte samples of 20 volunteers. While both extraction and methanolysis are pivotal steps, our final consideration is to analyze sphingolipids and sphingoid bases under different chromatographic conditions, minding the interferences