HPLC–(Q)-TOF-MS-Based Study of Plasma Metabolic Profile Differences Associated with Age in Pediatric Population Using an Animal Model

[EN] A deep knowledge about the biological development of children is essential for appropriate drug administration and dosage in pediatrics. In this sense, the best approximation to study organ maturation is the analysis of tissue samples, but it requires invasive methods. For this reason, surrogate matrices should be explored. Among them, plasma emerges as a potential alternative since it represents a snapshot of global organ metabolism. In this work, plasma metabolic profiles from piglets of different ages (newborns, infants, and children) obtained by HPLC–(Q)-TOF-MS at positive and negative ionization modes were studied. Improved clustering within groups was achieved using multiblock principal component analysis compared to classical principal component analysis. Furthermore, the separation observed among groups was better resolved by using partial least squares-discriminant analysis, which was validated by bootstrapping and permutation testing. Thanks to univariate analysis, 13 metabolites in positive and 21 in negative ionization modes were found to be significant to discriminate the three groups of piglets. From these features, an acylcarnitine and eight glycerophospholipids were annotated and identified as metabolites of interest. The findings indicate that there is a relevant change with age in lipid metabolism in which lysophosphatidylcholines and lysophoshatidylethanolamines play an important role.This research was funded by UPV/EHU (Project GIU16/04) and the Spanish Ministry of Economy and Competitiveness (Project CTQ2013-46179-R)

A novel SPE-UHPLC-DAD method for the determination of fumagillin produced by Aspergillus fumigatus in cell culture media

[EN]Fumagillin is a biomolecule produced by Aspergillus fumigatus that is gaining relevance due to its connection with invasive aspergillosis. The determination of this molecule might help to understand the propagation of this disease and study its use as a potential biomarker. In spite of the interest of fumagillin in microbiological research, no quantitative method has been developed so far for its determination in cell culture media. In this work, the first validated method for the quantitative analysis of fumagillin in RPMI-1640 is presented. The sample treatment consists of a mixed-mode anion exchange Solid Phase Extraction that effectively removes potential interferences and offered a recovery of 83 ± 7%. The analysis was carried out by Ultra High Performance Liquid Chromatography coupled to Diode Array Detection at 336 nm. The method fulfilled the validation criteria established by EMA and FDA guidelines for bioanalysis (selectivity, carry over, linearity, accuracy, precision, dilution integrity and stability) and offers a limit of quantitation (25 μg·L−1) suitable for its intended use. Indeed, the method was satisfactorily applied to the quantification of the fumagillin produced by three strains of Aspergillus fumigatus with different toxin production capacity.This research was funded by University of the Basque Country (UPV/EHU) (project GIU19/068 and project COLAB20/11) and by Basque Government (grant number IT1362-19). XGP was funded by Basque Government

Study of antifungal agent caspofungin adsorption to laboratory materials

[EN] Treatment of invasive fungal infections with Caspofungin is used as the first-line antifungal agents. The minimum inhibitory concentration value is a test which indicates the degree of sensitivity of a strain regarding a drug. However, no value of minimum inhibitory concentration for caspofungin is available because very variable value is obtained. In this work, we study the link with the adsorption phenomenon of CSF previously described in literature and the lack of minimum inhibitory concentration value. A systematic study of the impact of different parameters on CSF adsorption is reported. The effect of the nature of container material, the aqueous solution pH and the organic solvent proportion was studied. In addition, the possibility of using a coating agent to minimize the adsorption was assayed and evaluated. Results obtained showed the importance of the material used during the manipulation of CSF. The use of acidic pH aqueous solution or the addition of acetonitrile or methanol proportions (50 % and 70 %, respectively) were found efficient to avoid adsorption of CSF on glassware material, which is the relevant strategy for analytical samples of caspofungin. The treatment of HPLC glass vials and 96-well plates with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane reduced the adsorption. The significant adsorption observed in this work especially with plastic materials, questions the results obtained before in different assays and explained the absence of MIC value.The authors thank University of the Basque Country (UPV/EHU) (Project GIU19/068 and Project COLAB20/11) and Basque Government (grant number IT1362-19) for financial support. B. Uribe thanks UPV/EHU for the pre-doctoral fellowship in co-supervision with the University of Bordeaux. X. Guruceaga thanks the Basque Government for his predoctoral grant

Sindrome metabolikoa: XXI. mendearen madarikazioa

Bizi-baldintzen etengabeko bilakaeran gaur egungo gizarteak bereganatu dituen hainbat ohituren ondorioz (dieta desorekatua eta ariketa fisiko eskasa besteak beste), esan daiteke eritasun kardiobasku larren intzidentzia goraka doala . Eritasun kardiobaskularren izenpean hainbat patologia topa ditzakegu (b ihotzekoa , istripu zerebrobaskularra .. . ) eta hauen atzean dauden arrisku-faktoreak desberdinak dira: hipertentsioa, kolesterola, diabetesa, obesitatea ... Arrisku-faktore hauek ez dira normalean banaka agertzen, eta elkarrekin konbinatzen dira sindrome merabolikoa delakoaren barruan. Sindrome metabolikoa kon trolatzeko bizimoduan aldaketa txikiak baina garrantzitsuak ezarri behar dira eta neurri horiek nahikoak ez direnean bakarrik hasi behar gara medikamentuak erabi ltzen. Arrisku-faktore bakoitzak tratamendu ezberdina behar duenez, beharrezkoa gertatzen da farmako ezberdinez osatu riko terapia kardiobaskular konbinarua delakoa. Orokorrean, terapia honetan, hipertentsioaren aurkako botikak erabiltzen dira presio arteriala jaisteko, hipolipemianteak gorputzeko gantz kontzentrazioak kontrolatzeko, eta bestetik intsulina edo diabetesaren aurkako botikak glukosa mailak murrizteko. Horretaz gain, koagulazioa eta agregazio plaketarioa saihesteko botikak erabiltzen dira, sarritan gertatzen den arterien buxadurak ekiditeko

Sindrome metabolikoa: XXI. mendearen madarikazioa

Bizi-baldintzen etengabeko bilakaeran gaur egungo gizarteak bereganatu dituen hainbat ohituren ondorioz (dieta desorekatua eta ariketa fisiko eskasa besteak beste), esan daiteke eritasun kardiobasku larren intzidentzia goraka doala . Eritasun kardiobaskularren izenpean hainbat patologia topa ditzakegu (b ihotzekoa , istripu zerebrobaskularra .. . ) eta hauen atzean dauden arrisku-faktoreak desberdinak dira: hipertentsioa, kolesterola, diabetesa, obesitatea ... Arrisku-faktore hauek ez dira normalean banaka agertzen, eta elkarrekin konbinatzen dira sindrome merabolikoa delakoaren barruan. Sindrome metabolikoa kon trolatzeko bizimoduan aldaketa txikiak baina garrantzitsuak ezarri behar dira eta neurri horiek nahikoak ez direnean bakarrik hasi behar gara medikamentuak erabi ltzen. Arrisku-faktore bakoitzak tratamendu ezberdina behar duenez, beharrezkoa gertatzen da farmako ezberdinez osatu riko terapia kardiobaskular konbinarua delakoa. Orokorrean, terapia honetan, hipertentsioaren aurkako botikak erabiltzen dira presio arteriala jaisteko, hipolipemianteak gorputzeko gantz kontzentrazioak kontrolatzeko, eta bestetik intsulina edo diabetesaren aurkako botikak glukosa mailak murrizteko. Horretaz gain, koagulazioa eta agregazio plaketarioa saihesteko botikak erabiltzen dira, sarritan gertatzen den arterien buxadurak ekiditeko

Aspergillus fumigatus Fumagillin Contributes to Host Cell Damage

The activity of fumagillin, a mycotoxin produced by Aspergillus fumigatus, has not been studied in depth. In this study, we used a commercial fumagillin on cultures of two cell types (A549 pneumocytes and RAW 264.7 macrophages). This toxin joins its target, MetAP2 protein, inside cells and, as a result, significantly reduces the electron chain activity, the migration, and the proliferation ability on the A549 cells, or affects the viability and proliferation ability of the RAW 264.7 macrophages. However, the toxin stimulates the germination and double branch hypha production of fungal cultures, pointing out an intrinsic resistant mechanism to fumagillin of fungal strains. In this study, we also used a fumagillin non-producer A. fumigatus strain (∆fmaA) as well as its complemented strain (∆fmaA::fmaA) and we tested the fumagillin secretion of the fungal strains using an Ultra High-Performance Liquid Chromatography (UHPLC) method. Furthermore, fumagillin seems to protect the fungus against phagocytosis in vitro, and during in vivo studies using infection of immunosuppressed mice, a lower fungal burden in the lungs of mice infected with the ∆fmaA mutant was demonstrated.This research was funded by the Basque Government: grant number IT1362-19. X.G. and S.C.-S. received a Ph.D. fellowship from the Basque Government; and U.P.-C. from the University of the Basque Country

Determination of drugs used in combined cardiovascular therapy

Cardiovascular diseases are nowadays the first cause of mortality worldwide, causing around the 30% of global deaths each year. The risk of suffering from cardiovascular illnesses is strongly related to some factors such as hypertension, high cholesterol levels, diabetes, obesity The combination of these different risk factors is known as metabolic syndrome and it is considered a pandemic due to the high prevalence worldwide. The pathology of the disorders implies a combined cardiovascular therapy with drugs which have different targets and mechanisms of action, to regulate each factor separately. The simultaneous analysis of these drugs turns interesting but it is a complex task since the determination of multiple substances with different physicochemical properties and physiological behavior is always a challenge for the analytical chemist. The complexity of the biological matrices and the difference in the expected concentrations of some analytes require the development of extremely sensitive and selective determination methods. The aim of this work is to fill the gap existing in this field of the drug analysis, developing analytical methods capable of quantifying the different drugs prescribed in combined cardiovascular therapy simultaneously. Liquid chromatography andem mass spectrometry (LCMS/MS) has been the technique of choice throughout the main part of this work, due to the high sensitivity and selectivity requirements