Optimization of sample extraction and injection-related parameters in HILIC performance for polar metabolite analysis. Application to the study of a model of pulmonary hypertension

Morán-Garrido, María; Muñoz-Escudero, Patricia; García‐Álvarez, Ana; García‐Lunar, Inés; Barbas, Coral; Sáiz, Jorge

doi:10.1016/j.chroma.2022.463626

Cited by 4 publications

(3 citation statements)

References 29 publications

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“…In the negative mode, we used a stacking of 8 μL of ACN in both leading and terminating bands. The gradient consisted of a mix of mobile phases A and B as described in 38 .…”

Section: Methodsmentioning

confidence: 99%

Early Leishmania infectivity depends on miR-372/373/520d family-mediated reprogramming of polyamines metabolism in THP-1-derived macrophages

Fernandes,

Muxel,

López-Gonzálvez

et al. 2024

Sci Rep

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Leishmania amazonensis is a protozoan that primarily causes cutaneous leishmaniasis in humans. The parasite relies on the amino acid arginine to survive within macrophages and establish infection, since it is a precursor for producing polyamines. On the other hand, arginine can be metabolized via nitric oxide synthase 2 (NOS2) to produce the microbicidal molecule nitric oxide (NO), although this mechanism does not apply to human macrophages since they lack NOS2 activity. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at posttranscriptional levels. Our previous work showed that mmu-miR-294 targets Nos2 favoring Leishmania survival in murine macrophages. Here, we demonstrate that human macrophages upregulate the hsa-miR-372, hsa-miR-373, and hsa-miR-520d, which present the same seed sequence as the murine mmu-miR-294. Inhibition of the miR-372 impaired Leishmania survival in THP-1 macrophages and the effect was further enhanced with combinatorial inhibition of the miR-372/373/520d family, pointing to a cooperative mechanism. However, this reduction in survival is not caused by miRNA-targeting of NOS2, since the seed-binding motif found in mice is not conserved in the human 3′UTR. Instead, we showed the miR-372/373/520d family targeting the macrophage’s main arginine transporter SLC7A2/CAT2 during infection. Arginine-related metabolism was markedly altered in response to infection and miRNA inhibition, as measured by Mass Spectrometry-based metabolomics. We found that Leishmania infection upregulates polyamines production in macrophages, as opposed to simultaneous inhibition of miR-372/373/520d, which decreased putrescine and spermine levels compared to the negative control. Overall, our study demonstrates miRNA-dependent modulation of polyamines production, establishing permissive conditions for intracellular parasite survival. Although the effector mechanisms causing host cell immunometabolic adaptations involve various parasite and host-derived signals, our findings suggest that the miR-372/373/520d family may represent a potential target for the development of new therapeutic strategies against cutaneous leishmaniasis.

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“…In the negative mode, we used a stacking of 8 μL of ACN in both leading and terminating bands. The gradient consisted of a mix of mobile phases A and B as described in 38 .…”

Section: Methodsmentioning

confidence: 99%

Early Leishmania infectivity depends on miR-372/373/520d family-mediated reprogramming of polyamines metabolism in THP-1-derived macrophages

Fernandes,

Muxel,

López-Gonzálvez

et al. 2024

Sci Rep

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“…Metabolomic analyses were performed in samples obtained from all animals at end of follow-up (month 8). Lipids for lipidomics analysis and polar compounds for analysis by hydrophilic interaction liquid chromatography (HILIC) were extracted from plasma using previously described protocols [ 15 ]. LC-based lipidomics and HILIC were performed with an Agilent 1290 Infinity II ultra-high performance liquid chromatography (UHPLC) system coupled either to an Agilent 6560 ion mobility quadrupole time-of-flight (IM-QTOF) mass spectrometer working in QTOF mode or to a 6545 QTOF mass spectrometer (Agilent Technologies Waldbronn, Germany).…”

Section: Methodsmentioning

confidence: 99%

Metabolic changes contribute to maladaptive right ventricular hypertrophy in pulmonary hypertension beyond pressure overload: an integrative imaging and omics investigation

García-Lunar,

Jorge,

Sáiz

et al. 2024

Basic Res Cardiol

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Right ventricular (RV) failure remains the strongest determinant of survival in pulmonary hypertension (PH). We aimed to identify relevant mechanisms, beyond pressure overload, associated with maladaptive RV hypertrophy in PH. To separate the effect of pressure overload from other potential mechanisms, we developed in pigs two experimental models of PH (M1, by pulmonary vein banding and M2, by aorto-pulmonary shunting) and compared them with a model of pure pressure overload (M3, pulmonary artery banding) and a sham-operated group. Animals were assessed at 1 and 8 months by right heart catheterization, cardiac magnetic resonance and blood sampling, and myocardial tissue was analyzed. Plasma unbiased proteomic and metabolomic data were compared among groups and integrated by an interaction network analysis. A total of 33 pigs completed follow-up (M1, n = 8; M2, n = 6; M3, n = 10; and M0, n = 9). M1 and M2 animals developed PH and reduced RV systolic function, whereas animals in M3 showed increased RV systolic pressure but maintained normal function. Significant plasma arginine and histidine deficiency and complement system activation were observed in both PH models (M1&M2), with additional alterations to taurine and purine pathways in M2. Changes in lipid metabolism were very remarkable, particularly the elevation of free fatty acids in M2. In the integrative analysis, arginine–histidine–purines deficiency, complement activation, and fatty acid accumulation were significantly associated with maladaptive RV hypertrophy. Our study integrating imaging and omics in large-animal experimental models demonstrates that, beyond pressure overload, metabolic alterations play a relevant role in RV dysfunction in PH.

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“…RPLC is employed in most untargeted studies, as it generates reproducible data for a large set of non- to moderately polar metabolites. Recently, however, with the introduction of reproducible and robust stationary phases, HILIC has gained popularity in metabolomics research [ 10 , 11 , 12 ]. HILIC provides complementary selectivity to RPLC, retaining moderately to highly polar analytes [ 13 ], and coupled with MS has been frequently used for studies of metabolites in human plasma [ 14 ], urine [ 15 ], bacterial extracts [ 16 ], and many other biological samples [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

Optimization Strategies for Mass Spectrometry-Based Untargeted Metabolomics Analysis of Small Polar Molecules in Human Plasma

Kaczmarek,

Zhang,

Buzhansky

et al. 2023

Metabolites

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The untargeted approach to mass spectrometry-based metabolomics has a wide potential to investigate health and disease states, identify new biomarkers for diseases, and elucidate metabolic pathways. All this holds great promise for many applications in biological and chemical research. However, the complexity of instrumental parameters on advanced hybrid mass spectrometers can make the optimization of the analytical method immensely challenging. Here, we report a strategy to optimize the selected settings of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for untargeted metabolomics studies of human plasma, as a sample matrix. Specifically, we evaluated the effects of the reconstitution solvent in the sample preparation procedure, the injection volume employed, and different mass spectrometry-related operating parameters including mass range, the number of data-dependent fragmentation scans, collision energy mode, duration of dynamic exclusion time, and mass resolution settings on the metabolomics data quality and output. This study highlights key instrumental variables influencing the detection of metabolites along with suggested settings for the IQ-X tribrid system and proposes a new methodological framework to ensure increased metabolome coverage.

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Optimization of sample extraction and injection-related parameters in HILIC performance for polar metabolite analysis. Application to the study of a model of pulmonary hypertension

Cited by 4 publications

References 29 publications

Early Leishmania infectivity depends on miR-372/373/520d family-mediated reprogramming of polyamines metabolism in THP-1-derived macrophages

Early Leishmania infectivity depends on miR-372/373/520d family-mediated reprogramming of polyamines metabolism in THP-1-derived macrophages

Metabolic changes contribute to maladaptive right ventricular hypertrophy in pulmonary hypertension beyond pressure overload: an integrative imaging and omics investigation

Optimization Strategies for Mass Spectrometry-Based Untargeted Metabolomics Analysis of Small Polar Molecules in Human Plasma

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