In-Vial Dual Extraction for Direct LC-MS Analysis of Plasma for Comprehensive and Highly Reproducible Metabolic Fingerprinting

Metabolic fingerprinting of biological tissues has become an important area of research, particularly in the biomarker discovery field. Methods have inherent analytical variation, and new approaches are necessary to ensure that the vast numbers of intact metabolites present in biofluids are detected. Here, we describe an in-vial dual extraction (IVDE) method and a direct injection method that shows the total number of features recovered to be over 4500 from a single 20 μL plasma aliquot. By applying a one-step extraction consisting of a lipophilic and hydrophilic layer within a single vial insert, we showed that analytical variation was decreased. This was achieved by reducing sample preparation stages including procedures of drying and transfers. The two phases in the vial, upper and lower, underwent HPLC-QTOF analysis on individually customized LC gradients in both positive and negative ionization modes. A 60 min lipid profiling HPLC-QTOF method for the lipophilic phase was specifically developed, enabling the separation and putative identification of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, and sterols. The aqueous phase of the extract underwent direct injection onto a 45 min gradient, enabling the detection of both polarities. The IVDE method was compared to two traditional extraction methods. The first method was a two-step ether evaporation and IPA resuspension, and the second method was a methanol precipitation typically used in fingerprinting studies. The IVDE provided a 378% increase in reproducible features when compared to evaporation and a 269% increase when compared to the precipitate and inject method. As a proof of concept, the method was applied to an animal model of diabetes. A 2-fold increase in discriminant metabolites was found when comparing diabetic and control rats with IVDE. These discriminant metabolites accounted for around 600 entities, out of which 388 were identified in available databases

In-vial dual extraction liquid chromatography coupled to mass spectrometry applied to streptozotocin-treated diabetic rats.:Tips and pitfalls of the method

The aim of metabolomics studies is the comprehensive and quantitative analysis of all metabolites in a cell, tissue or organism. This approach requires sample preparation methods to be fast, reproducible and able to extract a wide range of analytes with different polarities, as well as analytical platforms able to detect the extracted metabolites. Recently, we have developed a one-step extraction method consisting of a lipophilic and hydrophilic layer within a single vial insert, in-vial dual extraction (IVDE). In order to check possible application of this method to real biological case, analysis of plasma samples obtained from three streptozotocin-induced diabetic and three control rats was performed. Analytical validity of the method was proved by the calculation (in quality control samples) of relative standard deviation (RSD) for detected metabolites. The percentage of metabolites with RSD < 30% was 93% for Fatty acyls, 80% for Glycerolipids, 93% for Glycerophospholipids, 68% for Sterol lipids, and 91% for Sphingolipids. IVDE allowed for selection of more than 600 different features discriminating two studied groups. For around 40% of these masses putative identification was possible. Adequate, with several considerations described within this paper, application of IVDE method enables wide metabolite coverage from a single 20 μL plasma aliquot. Within the features putatively identified, glycerolipids and glycerophospholipids arose as the most important groups of compounds discriminating diabetic rats from controls. All discriminating metabolites give an idea of the large metabolic differences that can be present in non-controlled type 1 diabetes

Plasma Metabolic Signature of Atherosclerosis Progression and Colchicine Treatment in Rabbits.

Balloon catheter endothelial denudation in New Zealand white rabbits fed high cholesterol diet is a validated atherosclerosis model. Well-characterized in terms of atherosclerosis induction and progression, the metabolic changes associated with the atherosclerosis progression remain indeterminate. Non-targeted metabolomics permits to develop such elucidation and allows to evaluate the metabolic consequences of colchicine treatment, an anti-inflammatory drug that could revert these changes. 16 rabbits underwent 18 weeks of atherosclerosis induction by diet and aortic denudation. Thereafter animals were randomly assigned to colchicine treatment or placebo for 18 weeks while on diet. Plasma samples were obtained before randomization and at 36 weeks. Multiplatform (GC/MS, CE/MS, RP-HPLC/MS) metabolomics was applied. Plasma fingerprints were pre-processed, and the resulting matrixes analyzed to unveil differentially expressed features. Different chemical annotation strategies were accomplished for those significant features. We found metabolites associated with either atherosclerosis progression, or colchicine treatment, or both. Atherosclerosis was profoundly associated with an increase in circulating bile acids. Most of the changes associated with sterol metabolism could not be reverted by colchicine treatment. However, the variations in lysine, tryptophan and cysteine metabolism among others, have shown new potential mechanisms of action of the drug, also related to atherosclerosis progression, but not previously described.M.A.I. received funding from Airbus Defense and Space through the CLX-2 program developed with Comando da Aeronáutica (COMAER) and the Brazilian Government. This work has been partially funded by the Ministerio de Ciencia, Innovación y Universidades of Spain (MICINN RTI2018-095166-B-I00), the Spanish Society of Cardiology (“Proyecto investigación traslacional” to BI), the Carlos III Institute of Health (ISCiii FIS-FEDER PI14-01427 to JM), the Ministerio de Economía, Industria y Competitividad (MINECO SAF2017-84494-C2-1R to JR-C), a project granted by the BBVA Foundation to JR-C. This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (MDM-2017-0720). The CNIC is supported by the ISCiii, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Metabolic impact of partial hepatectomy in the non-alcoholic steatohepatitis animal model of methionine-choline deficient diet

In the liver, obesity is often manifested by the clinical disorder of the Non-Alcoholic Fatty Liver Disease (NAFLD). A proportion of NAFLD patients develop hepatic inflammation, known as Non-Alcoholic Steatohepatitis (NASH), which can end up in cirrhosis, or Hepatocellular Carcinoma (HCC). In this scenario, partial hepatectomy (PH) is an alternative to promote liver regeneration. However, as liver regeneration is impaired in NASH patients, more knowledge about its metabolic condition is needed to improve the regenerative response of the liver in this pathological condition. Although extensively employed, the panoply of molecular alterations involved in the regenerative response of the liver after partial hepatectomy PH is far from being fully characterized. Metabolic fingerprinting (metabolomics) is a powerful tool to help in the elucidation of complex metabolic networks, by means of a blind, naïve approach to study which metabolic nodes (metabolites) show the biggest variations between conditions. The objective of the present study was to gain deeper knowledge about the metabolic processes involved in the NASH animal model, and particularly in the effect of PH by using metabolomics. For achieving such information, twelve 8-week-old male C57BL/6 J mice, fed commercial chow (control diet) or methionine and choline-Deficient diet (MCD) for three weeks were subjected to PH and sacrificed 2 weeks later. Livers were removed and submitted to metabolic profiling analysis through RP-LC/MS (qTOF), GC/MS (qTOF) and CE/MS(TOF). More than 3000 different features were detected and repeated measurements one-way ANOVA analysis was performed to unveil significant features. MCD diet induced changes (p < 0.05) in 46% of the detected features, whereas PH provoked significant changes in 85% of them. Most of the changes were detected through LC/MS and were associated to lipid metabolism. However, changes of metabolites virtually related to other metabolic routes (amino acids, carbohydrates, nucleotides) were found altered and detected by CE/MS and GC/MS. The changes associated to PH show a similar trend regardless of the diet, but in the context of the diet deficient in methionine and choline we have found results that point to a different ratio glycolysis/tricarboxylic acid cycle. Moreover, in the NASH model, the regeneration of the liver structures occurs at the expense of an increased phosphatidylethanolamines/phosphatidylcholines ratio.This work has been partially funded by the Ministerio de Ciencia, Innovación y Universidades of Spain (MICINN RTI2018-095166-B-I00, MICINN/FEDER RTI2018-094052-B-100) and Comunidad de Madrid of Spain (S2017/BMD-3684 MOIR2-CM). A.M.V. received funding from CIBERdem (ISCIII, Spain)."Peer reviewe

In-Vial Dual Extraction for Direct LC-MS Analysis of Plasma for Comprehensive and Highly Reproducible Metabolic Fingerprinting.

Metabolic fingerprinting of biological tissues has become an important area of research, particularly in the biomarker discovery field. Methods have inherent analytical variation, and new approaches are necessary to ensure that the vast numbers of intact metabolites present in biofluids are detected. Here, we describe an in-vial dual extraction (IVDE) method and a direct injection method that shows the total number of features recovered to be over 4500 from a single 20 μL plasma aliquot. By applying a one-step extraction consisting of a lipophilic and hydrophilic layer within a single vial insert, we showed that analytical variation was decreased. This was achieved by reducing sample preparation stages including procedures of drying and transfers. The two phases in the vial, upper and lower, underwent HPLC-QTOF analysis on individually customized LC gradients in both positive and negative ionization modes. A 60 min lipid profiling HPLC-QTOF method for the lipophilic phase was specifically developed, enabling the separation and putative identification of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, and sterols. The aqueous phase of the extract underwent direct injection onto a 45 min gradient, enabling the detection of both polarities. The IVDE method was compared to two traditional extraction methods. The first method was a two-step ether evaporation and IPA resuspension, and the second method was a methanol precipitation typically used in fingerprinting studies. The IVDE provided a 378% increase in reproducible features when compared to evaporation and a 269% increase when compared to the precipitate and inject method. As a proof of concept, the method was applied to an animal model of diabetes. A 2-fold increase in discriminant metabolites was found when comparing diabetic and control rats with IVDE. These discriminant metabolites accounted for around 600 entities, out of which 388 were identified in available databases

A complex interplay between sphingolipid and sterol metabolism revealed by perturbations to the Leishmania metabolome caused by miltefosine

With the World Health Organization reporting over 30,000 deaths and 200-400,000 new cases annually, visceral Leishmaniasis is a serious disease affecting some of the world's poorest people. As drug resistance continues to rise, there is a huge unmet need to improve treatment. Miltefosine remains one of the main treatments for Leishmaniasis, yet its mode of action (MoA) is still unknown. Understanding the MoA of this drug and parasite response to treatment could help pave the way for new, more successful treatments for Leishmaniasis. A novel method has been devised to study the metabolome and lipidome of Leishmania donovani axenic amastigotes treated with miltefosine. Miltefosine caused a dramatic decrease in many membrane phospholipids (PLs), in addition to amino acid pools, while sphingolipids (SLs) and sterols increased. Leishmania major promastigotes devoid of SL biosynthesis through loss of the serine palmitoyl transferase gene (ΔLCB2) were 3-fold less sensitive to miltefosine than WT parasites. Changes in the metabolome and lipidome of miltefosine treated L. major mirrored those of L. donovani. A lack of SLs in the ΔLCB2 was matched by substantial alterations in sterol content. Together these data indicate that SLs and ergosterol are important for miltefosine sensitivity and perhaps, MoA

A single in-vial dual extraction strategy for the simultaneous lipidomics and proteomics analysis of HDL and LDL fractions

A single in-vial dual extraction (IVDE) procedure for the subsequent analysis of lipids and proteins in the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions derived from the same biological sample is presented. On the basis of methyl-tert-butyl ether (MTBE) extraction, IVDE leads to the formation of three phases: a protein pellet at the bottom, an aqueous phase with polar compounds, and an ether phase with lipophilic compounds. After sample extraction, performed within a high-performance liquid chromatography vial insert, the ether phase was directly injected for lipid fingerprinting, while the protein pellet, after evaporation of the remaining sample, was used for proteomics analysis. Human HDL and LDL isolates were used to test the suitability of the IVDE methodology for lipid and protein analysis from a single sample in terms of data quality and matching composition to that of HDL and LDL. Subsequently, HDL and LDL fractions isolated from ApoE-KO and wild-type mice were used to validate the capacity of IVDE for revealing changes in lipid and protein abundance. Results indicate that IVDE can be successfully used for the subsequent analysis of lipids and proteins with the advantages of time saving, simplicity, and reduced sample amount