Quantitative plasma profiling by 1H NMR-based metabolomics: impact of sample treatment

Introduction: There is evidence that sample treatment of blood-based biosamples may affect integral signals in nuclear magnetic resonance-based metabolomics. The presence of macromolecules in plasma/serum samples makes investigating low-molecular-weight metabolites challenging. It is particularly relevant in the targeted approach, in which absolute concentrations of selected metabolites are often quantified based on the area of integral signals. Since there are a few treatments of plasma/serum samples for quantitative analysis without a universally accepted method, this topic remains of interest for future research. Methods: In this work, targeted metabolomic profiling of 43 metabolites was performed on pooled plasma to compare four methodologies consisting of Carr-Purcell-Meiboom-Gill (CPMG) editing, ultrafiltration, protein precipitation with methanol, and glycerophospholipid solid-phase extraction (g-SPE) for phospholipid removal; prior to NMR metabolomics analysis. The effect of the sample treatments on the metabolite concentrations was evaluated using a permutation test of multiclass and pairwise Fisher scores. Results: Results showed that methanol precipitation and ultrafiltration had a higher number of metabolites with coefficient of variation (CV) values above 20%. G-SPE and CPMG editing demonstrated better precision for most of the metabolites analyzed. However, differential quantification performance between procedures were metabolite-dependent. For example, pairwise comparisons showed that methanol precipitation and CPMG editing were suitable for quantifying citrate, while g-SPE showed better results for 2-hydroxybutyrate and tryptophan. Discussion: There are alterations in the absolute concentration of various metabolites that are dependent on the procedure. Considering these alterations is essential before proceeding with the quantification of treatment-sensitive metabolites in biological samples for improving biomarker discovery and biological interpretations. The study demonstrated that g-SPE and CPMG editing are effective methods for removing proteins and phospholipids from plasma samples for quantitative NMR analysis of metabolites. However, careful consideration should be given to the specific metabolites of interest and their susceptibility to the sample treatment procedures. These findings contribute to the development of optimized sample preparation protocols for metabolomics studies using NMR spectroscop

Prenatal greenspace exposure and cord blood cortisol levels : A cross-sectional study in a middle-income country

Exposure to greenspace has been associated with reduced stress; however, the available evidence on such an association for the fetus is still very scarce. We, for the first time, investigated the association between maternal greenspace exposure and the level of cortisol, a stress hormone, in the cord blood. Our study was based on a cohort of 150 pregnant women in Sabzevar, Iran (2018). We comprehensively assessed greenspace exposure for each participant through (i) residential surrounding greenspace (using two satellite-derived vegetation indices), (ii) residential proximity to green spaces, (iii) maternal visual access to greenspace, (iv) use of public and private green spaces, (v) having a private garden, and (vi) the number of plant pots at home. Linear regression models were developed to assess the association of each indicator of greenspace exposure with cord blood cortisol levels, controlled for the relevant covariates. We observed that a higher residential surrounding greenspace (100 m buffer), having a window with greenspace view, window greenspace coverage of more than 50%, frequently looking at greenspace through window, residential proximity to large green spaces, and more time spent in green spaces were associated with lower cortisol levels in the cord blood. The findings for residential surrounding greenspace at 300 m and 500 m buffers, residential proximity to any green space regardless of its size, having a private garden, and number of plant pots at home were not conclusive. While about one-third of the association between residential surrounding greenspace (100 m buffer) could be mediated through reduction in exposure to air pollution, we did not observe any strong evidence for such a mediatory role for the visual access to greenspace. The findings stratified for parental education and housing type showed mixed patterns. Our findings suggest that more greenspace exposure might reduce cortisol level in the cord blood

ABCG2 transporter plays a key role in the biodistribution of melatonin and its main metabolites

[EN] The ATP-binding cassette G2 (ABCG2) is an efflux transporter expressed in the apical membrane of cells from a large number of tissues, directly affecting bioavailability, tissue accumulation, and secretion into milk of both xenobiotics and endogenous compounds. The aim of this work was to characterize the role of ABCG2 in the systemic distribution and secretion into milk of melatonin and its main metabolites, 6-hydroxymelatonin, and 6-sulfatoxymelatonin. For this purpose, we first showed that these three molecules are transported by this transporter using in vitro transepithelial assays with MDCK-II polarized cells transduced with different species variants of ABCG2. Second, we tested the in vivo effect of murine Abcg2 in the systemic distribution of melatonin and its metabolites using wild-type and Abcg2−/− mice. Our results show that after oral administration of melatonin, the plasma concentration of melatonin metabolites in Abcg2−/− mice was between 1.5 and 6-fold higher compared to the wild-type mice. We also evaluated in these animals differences in tissue accumulation of melatonin metabolites. The most relevant differences between both types of mice were found for small intestine and kidney (>sixfold increase for 6-sulfatoxymelatonin in Abcg2−/− mice). Finally, melatonin secretion into milk was also affected by the murine Abcg2 transporter, with a twofold higher milk concentration in wild-type compared with Abcg2−/− lactating female mice. In addition, melatonin metabolites showed a higher milk-to-plasma ratio in wild-type mice. Overall, our results show that the ABCG2 transporter plays a critical role in the biodistribution of melatonin and its main metabolites, thereby potentially affecting their biological and therapeutic activity.SIPublicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCL

Characterization of Domiphen Bromide as a New Fast-Acting Antiplasmodial Agent Inhibiting the Apicoplastidic Methyl Erythritol Phosphate Pathway

The evolution of resistance by the malaria parasite to artemisinin, the key component of the combination therapy strategies that are at the core of current antimalarial treatments, calls for the urgent identification of new fast-acting antimalarials. The apicoplast organelle is a preferred target of antimalarial drugs because it contains biochemical processes absent from the human host. Fosmidomycin is the only drug in clinical trials targeting the apicoplast, where it inhibits the methyl erythritol phosphate (MEP) pathway. Here, we characterized the antiplasmodial activity of domiphen bromide (DB), another MEP pathway inhibitor with a rapid mode of action that arrests the in vitro growth of Plasmodium falciparum at the early trophozoite stage. Metabolomic analysis of the MEP pathway and Krebs cycle intermediates in 20 mu M DB-treated parasites suggested a rapid activation of glycolysis with a concomitant decrease in mitochondrial activity, consistent with a rapid killing of the pathogen. These results present DB as a model compound for the development of new, potentially interesting drugs for future antimalarial combination therapies

2-picolylamine derivatization for high sensitivity detection of abscisic acid in apicomplexan blood-infecting parasites

We have developed a new liquid chromatography-electrospray ionization tandem mass spectrometry methodology based on 2-picolylamine derivatization and positive ion mode detection for abscisic acid (ABA) identification. The selected reaction leads to the formation of an amide derivative which contains a highly active pyridyl group. The enhanced ionization allows for a 700-fold increase over commonly monitored unmodified ABA, which in turn leads to excellent limits of detection and quantification values of 0.03 and 0.15ngmL-1, respectively. This method has been validated in the highly complex matrix of a red blood cell extract. In spite of the high sensitivity achieved, ABA could not be detected in Plasmodium falciparum-infected red blood cells, suggesting that, if present, it will be found either in ultratrace amounts or as brief bursts at defined time points within the intraerythrocytic cycle and/or in the form of a biosynthetic analogue

Maternal separation increases alcohol-drinking behaviour and reduces endocannabinoid levels in the mouse striatum and prefrontal cortex

Altres ajuts: This study was supported by Plan Nacional sobre Drogas (2014/020) i UE Medbioinformatic project (grant number 634143).Childhood adversity is associated with an increased risk of mood, anxiety and substance use disorders. Maternal separation is a reliable rodent model of early life adversity that leads to depression-like symptoms, which may increase the vulnerability to alcohol consumption during adolescence. However, the specific alterations in the pattern of alcohol consumption induced by maternal separation and the underlying molecular mechanisms are still unclear. The purpose of this study is to evaluate the long-term effects of maternal separation with early weaning (MSEW) on emotional and social behaviour, alcohol rewarding properties, and alcohol consumption, abstinence and relapse in adolescent male C57BL/6 mice. In addition, endocannabinoid and monoamine levels were analysed in discrete brain areas. Results showed that MSEW mice presented emotional alterations related to depressive-like behaviour and modified endocannabinoid levels in the striatum and the prefrontal cortex. MSEW mice also showed impairments in alcohol-induced conditioned place preference and higher alcohol intake in a model of binge drinking. Moreover, MSEW animals displayed a higher propensity to relapse in the two-bottle choice paradigm following a period of alcohol abstinence associated with reduced monoamine levels in the striatum. Such results indicate that exposure to early life stress increased the vulnerability to alcohol binge-drinking during adolescence, which may be partially explained by decreased sensitivity to alcohol rewarding properties and the ability to potentiate alcohol intake following a period of abstinence

Analysis of the interaction between tryptophan-related compounds and ATP-binding cassette transporter G2 (ABCG2) using targeted metabolomics

ATP-binding cassette transporter G2 (ABCG2) is involved in the secretion of several compounds in milk. The in vitro and in vivo interactions between tryptophan-related compounds and ABCG2 were investigated. The tryptophan metabolome was determined by liquid chromatography-tandem mass spectrometry in milk and plasma from wild-type and Abcg2-/- mice as well as dairy cows carrying the ABCG2 Y581S polymorphism (Y/S) and noncarrier animals (Y/Y). The milk-to-plasma ratios of tryptophan, kynurenic acid, kynurenine, anthranilic acid, and xanthurenic acid were higher in wild-type mice than in Abcg2-/- mice. The ratio was 2-fold higher in Y/S than in Y/Y cows for kynurenine. In vitro transport assays confirmed that some of these compounds were in vitro substrates of the transporter and validated the differences observed between the two variants of the bovine protein. These findings show that the secretion of metabolites belonging to the kynurenine pathway into milk is mediated by ABCG2.SIThis study was supported by the research projects AGL2015-65626-R (MINECO/FEDER, UE) and RTI2018-100903-B-I00 (AEI/FEDER, UE), predoctoral grants from the Ministry of Economy, Industry, and Competitiveness (BES-2016-077235 grant to AMGL), and grants from the Spanish Ministry of Education, Culture, and Sport (FPU14/05131 grant to DGM). Funding was also obtained from a research contract for OJP from the Spanish Health Institute Carlos III (CPII16/00027

Evaluation of metabolic changes in acute intermittent porphyria patients by targeted metabolomics

Acute intermittent porphyria (AIP) is an inherited rare hepatic disorder due to mutations within the hydroxymethylbilane gene. AIP patients with active disease overproduce aminolevulinic acid (ALA) and porphobilinogen (PBG) in the liver which are exported inducing severe neurological attacks. Different hepatic metabolic abnormalities have been described to be associated with this condition. The goal of this research was to explore the metabolome of symptomatic AIP patients by state-of-the art liquid chromatography-tandem mass spectrometry (LC-MS/MS). A case versus control study including 18 symptomatic AIP patients and 33 healthy controls was performed. Plasmatic levels of 51 metabolites and 16 ratios belonging to four metabolic pathways were determined. The results showed that the AIP patients presented significant changes in the two main areas of the metabolome under study: (a) the tryptophan/kynurenine pathway with an increase of tryptophan in plasma together with increase of the kynurenine/tryptophan ratio; and (b) changes in the tricarboxylic acid cycle (TCA) including increase of succinic acid and decrease of the fumaric acid/succinic acid ratio. We performed a complementary in vitro study adding ALA to hepatocytes media that showed some of the effects on the TCA cycle were parallel to those observed in vivo. Our study confirms in plasma previous results obtained in urine showing that AIP patients present a moderate increase of the kynurenine/tryptophan ratio possibly associated with inflammation. In addition, it also reports changes in the mitochondrial TCA cycle that, despite requiring further research, could be associated with an energy misbalance due to sustained overproduction of heme-precursors in the liver.This research was funded by Instituto de Salud Carlos III FEDER (grant number PI14/00147), Generalitat de Catalunya (research team grant number 2014SGR692) and Spanish Health National System (contract number CPII16/00027 for Oscar J Pozo).Peer ReviewedPostprint (published version

Quantitative Estimation of Black Carbon in the Glacier Ampay-Apurimac

The presence of light-absorbing particles, including black carbon in glaciers leads to a reduction in albedo (light reflection), leading to further melting of snow ice, increased amount of shortwave solar radiation and leads to the glacier. The objective has been to determine the variation in the temporal space of black carbon, the amount of light-absorbing particles and the decrease of albedo on the surface of the Ampay glacier. 10 snow samples were selected at various locations on the glacier during 2017. The light absorption heating method has been applied to measure light-absorbing particles, a technique that measures the temperature increase of the particle charge in a filter applying visible light that estimates the amount of light energy. The results show in terms of effective black carbon, the highest of 65,224 nanograms of black carbon per gram of water in the month of October and the minimum value of 20,941 nanograms of black carbon per gram of water in the month of February, typically associated with the rain. The energy absorbed by the light-absorbing particles in the Ampay glacier, the highest corresponds to the month of November with 8,952.92 J s/m2 and lowest in February with 2,747.26 J s/m2. In April, the amount of snow melted due to light-absorbing particles has been approximately 13.57 kg/m2. Melting has increased considerably in other months with the largest melting, with a value of 26.65 kg/m2, almost 7.0 kW/m2 of snow turned into water in the month of November. It is concluded that the technique of the Light absorption heating method is adequate because it is optimal for the achievement of the research objectives, it is economical, effective and has allowed quantifying light-absorbing particles in snow