Posts classified under: Aside

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

February 2019.

Torquato P, Giusepponi D, Alisi A, Galarini R, Bartolini D, Piroddi M, Goracci L, Di Veroli A, Cruciani G, Crudele A, Nobili V, Galli F. 

Abstract

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 biopsyproven 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 untargeted 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. 

Delving into the Polar Lipidome by Optimized Chromatographic Separation, High-Resolution Mass Spectrometry, and Comprehensive Identification with Lipostar: Microalgae as Case Study

October 2018.

 La Barbera G, Antonelli M, Cavaliere C, Cruciani G, Goracci L, Montone CM, Piovesana S, Laganà A, Capriotti AL

Abstract

The work describes the chromatographic separation optimization of polar lipids on Kinetex-EVO, particularly focusing on sulfolipids in spirulina microalgae (Arthrospira platensis). Gradient shape and mobile phase modifiers (pH and buffer) were tested on lipid standards. Different conditions were evaluated and resolution, peak capacity and peak shape calculated both in negative mode, for sulfolipids and phospholipids, and in positive mode, for glycolipids. A high confidence lipid identification strategy was also applied. In collaboration with software creators and developers, Lipostar was implemented to improve the identification of phosphoglycerolipids and to allow the  identification of glycosylmonoradyl and glycosyldiradyl-glycerols classes, the last being the main focus of this work. By this approach, an untargeted screening also for searching lipids not yet reported in the literature could be accomplished. The optimized chromatographic conditions and database search were tested for lipid identification first on the standard mixture, then on the polar lipid extract of spirulina microalgae, for which 205 lipids were identified. 

Computational solutions in redox lipidomics – Current strategies and future perspectives

November 2019.

Ni Z, Goracci L, Cruciani G, Fedorova M

Abstract

The high chemical diversity of lipids allows them to perform multiple biological functions ranging from servingas structural building blocks of biological membranes to regulation of metabolism and signal transduction. In addition to the native lipidome, lipid species derived from enzymatic and non-enzymatic modifications (the epilipidome) make the overall picture even more complex, as their functions are still largely unknown. Oxidized lipids represent the fraction of epilipidome which has attracted high scientific attention due to their apparent involvement in the onset and development of numerous human disorders.

Development of high-throughput analytical methods such as liquid chromatography coupled on-line to mass spectrometry provides the possibility to address epilipidome diversity in complex biological samples. However, the main bottleneck of redox lipidomics, the branch of lipidomics dealing with the characterization of oxidized lipids, remains the lack of optimal computational tools for robust, accurate and specific identification of already discovered and yet unknown modified lipids. Here we discuss the main principles of high-throughput identification of lipids and their modified forms and review the main software tools currently available in redox lipidomics. Different levels of confidence for software assisted identification of redox lipidome are defined and necessary steps toward optimal computational solutions are proposed. 

Role of mitochondria and cardiolipins in growth inhibition of breast cancer cells by retinoic acid

October 2019

Terao M, Goracci L, Celestini V, Kurosaki M, Bolis M, Di Veroli A, Vallerga, A, Fratelli M, Lupi M, Corbelli A, Fiordaliso F, Gianni M, Paroni G, Zanetti A, Cruciani G, Garattini E.

Abstract

Background

All-trans-retinoic-acid (ATRA) is a promising agent in the prevention/treatment of breast-cancer. There is growing evidence that reprogramming of cellular lipid metabolism contributes to malignant transformation and progression. Lipid metabolism is implicated in cell differentiation and metastatic colonization, and it is involved in the mechanisms of sensitivity/resistance to different anti-tumor agents. The role played by lipids in the anti-tumor activity of ATRA has never been studied. 

Methods

We used 16 breast cancer cell-lines whose degree of sensitivity to the anti-proliferative action of ATRA is known. We implemented a non-oriented mass-spectrometry based approach to define the lipidomic profiles of each cell-line grown under basal conditions and following treatment with ATRA. To complement the lipidomic data, untreated and retinoid treated cell-lines were also subjected to RNA-sequencing to define the perturbations afforded by ATRA on the whole-genome gene-expression profiles. The number and functional activity of mitochondria were determined in selected ATRA-sensitive and -resistant cell-lines. Bio-computing approaches were used to analyze the high-throughput lipidomic and transcriptomic data. 

Results

ATRA perturbs the homeostasis of numerous lipids and the most relevant effects are observed on cardiolipins, which are located in the mitochondrial inner membranes and play a role in oxidative phosphorylation. ATRA reduces the amounts of cardiolipins, and the effect is associated with the growth-inhibitory activity of the retinoid. Down-regulation of cardiolipins is due to a reduction of mitochondria, which is caused by an ATRA-dependent decrease in the expression of nuclear genes encoding mitochondrial proteins. This demonstrates that ATRA anti-tumor activity is due to a decrease in the amounts of mitochondria causing deficits in the respiration/energy-balance of breast-cancer cells. 

Conclusions

The observation that ATRA anti-proliferative activity is caused by a reduction in the respiration and energy balance of the tumor cells has important ramifications for the therapeutic action of ATRA in breast cancer. The study may open the way to the development of rational therapeutic combinations based on the use of ATRA and anti-tumor agents targeting the mitochondria. 

An innovative algorithm to elucidate the structure of unknown compounds using tandem Mass Spectrometry and NMR data

65th ASMS Conference on Mass Spectrometry and Allied Topics, Indianapolis, IN (United States of America) 04 June 2017

Abstract

The interpretation of data obtained by tandem mass spectrometry is usually the bottleneck in different areas. This process becomes more complicated if the scientist does not have any clue about the structure of the analyzed compound. Until now, several algorithms have been developed to make easier the structural determination of the MS/MS data. Unfortunately, most of them use a database of interpreted MS/MS spectra where the input data is queried, which can reduce the number of potential results to those ones contained in the original dataset.

The algorithm developed and presented here makes the difference between other software in the origin of the initial dataset where the input MS/MS data is looked into. Methods The presented methodology is composed by three parts. The creation of the database is the first one. Users can choose a set of compounds from their own data or take them from an external database. Those compounds will be fragmented and stored on the database individually. In the second part, mz values from the input MS/MS are queried on the database and used to build a set of candidates by its rational combination. In the last part of the code, all the candidates are fragmented and compared with the peaks of the input MS/MS spectra. To decrease the number results, the NMR spectra of all of them is predicted and compared with the experimental NMR data of unknown compound. Preliminary Data The algorithm has been developed and successfully tested using a small set of compounds from the pharmacological area. One of them is 10P-909 (PubChem CID: 1480036; IUPAC name: 2-chloro-5-[[4-[3-(trifluoromethyl) phenyl] piperazin-1-yl]methyl]-1,3-thiazole).

The procedure to elucidate this benchmarking compound will be described in the following lines. The first step we made was the creation of the database. As a set of compounds, we use 500,000 structures from PubChem. Then, those compounds were fragmented using an in-house code, generating a final set of 6 million of independent fragments. Next, the algorithm was feed with the required parameters: the mz of the unknown structure, the tolerance given to this mz value, the ion mode, the adduct type and the MS/MS data. In this case study, the mz of the unknown compound is 362.0703, was acquired with positive ion mode and its adduct type is [M+H]+. Tolerance was set to 3 ppm because of the quality of the acquisition.

The original MS/MS input contains a total of 18 peaks, reduced to the half after removing isotopes. The mz data from the input culminates in a total of 6492 fragments from the database. The rational combination of them yields to up to 6500 solutions. To clean up the amount of solutions, each one was fragmented and later compared with the original MS/MS data. Close to 2000 results match with the 9 peaks of the original spectra.

Then, to obtain a most accurate result, both 1H and 13C NMR spectra of the best matched structures were predicted by an in house program and latter compared with the NMR data from the unknown structure. In our case, the 1H, 13C NMR and COSY spectra of the original unknown help us to find the structure of the 10P-909 on the first position of the ranking. Novel Aspect This algorithm operates with real fragments instead of using existing MS/MS spectra to predict the structure of the unknown compound.