Targeted profiling of circulating and hepatic bile acids in human, mouse, and rat using a UPLC-MRM-MS-validated method

Bile acids (BAs) are a group of chemically related steroids recognized as regulatory molecules whose profiles can change in different physio-pathological situations. We have developed a sensitive, fast, and reproducible ultraperformance liquid chromatography/multiple reaction monitoring/mass spectrometry method to determine the tissue and sera BA profiles in different species (human, rat, and mouse) by quantifying 31 major and minor BA species in a single 21-min run. The method has been validated according to FDA guidelines, and it generally provides good results in terms of intra- and interday precision (less than 8.6% and 16.0%, respectively), accuracy (relative error measurement between -11.9% and 8.6%), and linearity (R(2) > 0.996 and dynamic ranges between two and four orders of magnitude), with limits of quantification between 2.5 and 20 nM. The new analytical approach was applied to determine BA concentrations in human, rat, and mouse serum and in liver tissue. Our comparative study confirmed and extended previous reports, showing marked interspecies differences in circulating and hepatic BA composition. The targeted analysis revealed the presence of unexpected minoritary BAs, such as tauro-alpha-Muricholic acid in human serum, thus allowing us to obtain a thorough profiling of human samples. Its great sensitivity, low sample requirements (25 μl of serum, 5 mg of tissue), and comprehensive capacity to profile a considerable number of BAs make the present method a good choice to study BA metabolism in physiological and pathological situations, particularly in toxicological studies.     

Analysis of intact phosphoinositides in biological samples

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P(3)], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [(32)P]inorganic phosphate or [(3)H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS(3). Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.     

Personalized Proteome Profiles of Healthy and Tumor Human Colon Organoids Reveal Both Individual Diversity and Basic Features of Colorectal Cancer

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Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids

Human cytochrome P450 (P450) 2W1 is still considered an "orphan" because its physiological function is not characterized. To identify its substrate specificity, the purified recombinant enzyme was incubated with colorectal cancer extracts for untargeted substrate searches using an LC/MS-based metabolomic and isotopic labeling approach. In addition to previously reported fatty acids, oleyl (18:1) lysophosphatidylcholine (LPC, lysolecithin) was identified as a substrate for P450 2W1. Other human P450 enzymes tested showed little activity with 18:1 LPC. In addition to the LPCs, P450 2W1 acted on a series of other lysophospholipids, including lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidylglycerol, lysophosphatidylethanolamine, and lysophosphatidic acid but not diacylphospholipids. P450 2W1 utilized sn-1 18:1 LPC as a substrate much more efficiently than the sn-2 isomer; we conclude that the sn-1 isomers of lysophospholipids are preferred substrates. Chiral analysis was performed on the 18:1 epoxidation products and showed enantio-selectivity for formation of (9R,10S) over (9R,10S). The kinetics and position specificities of P450 2W1-catalyzed oxygenation of lysophospholipids (16:0 LPC and 18:1 LPC) and fatty acids (C16:0 and C18:1) were also determined. Epoxidation and hydroxylation of 18:1 LPC are considerably more efficient than for the C18:1 free fatty acid.     

A platform for efficient establishment and drug-response profiling of high-grade serous ovarian cancer organoids

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Profiling of phenolic glycosidic conjugates in leaves of Arabidopsis thaliana using LC/MS

Profiling of plant secondary metabolites is still a very difficult task. Liquid chromatography (LC) or capillary electrophoresis hyphenated with different kinds of detectors are methods of choice for analysis of polar, thermo labile compounds with high molecular masses. We demonstrate the applicability of LC combined with UV diode array or/and mass spectrometric detectors for the unambiguous identification and quantification of flavonoid conjugates isolated from Arabidopsis thaliana leaves of different genotypes and grown in different environmental conditions. During LC/UV/MS/MS analyses we were able to identify tetra-, tri-, and di-glycosides of kaempferol, quercetin and isorhamnetin. Based on our results we can conclude that due to the co-elution of different chemical compounds in reversed phase HPLC systems the application of UV detectors does not allow to precisely profile all flavonoid conjugates existing in A. thaliana genotypes. Using MS detection it was possible to unambiguously recognize the glycosylation patterns of the aglycones. However, from the mass spectra we could not conclude neither the anomeric form of the C-1 carbon atoms of sugar moieties in glycosidic bonds between sugars or sugar and aglycone nor the position of the second carbon involved in disaccharides. The applicability of collision induced dissociation techniques (CID MS/MS) for structural analyses of the studied group of plant secondary metabolites with two types of analyzers (triple quadrupole or ion trap) was demonstrated.     

Sensitive profiling of chemically diverse bioactive lipids

Here, we present an improved method for sensitive profiling of lipids in a single high-performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry experiment. The approach consists of i) sensitive isocratic elution, which takes advantage of C18 column material that is resistant to increased pH values induced by piperidine, ii) chemometric alignment of mass spectra followed by differential analysis of ion intensities, and iii) semiquantitative analysis of extracted ion chromatograms of interest. A key advantage of this method is its wide applicability to extracts that harbor lipids of considerable chemical complexity. The method allows qualitative and semiquantitative analysis of fatty acyls, glycerophospholipids (such as glycerophosphatidylinositols, glycerophosphatidylserines, and glycerophosphatidylcholines in brain extracts), phosphatidylinositol mannosides, acylated glycerophospholipids, sphingolipids (including ceramides and gangliosides in brain extracts), and, for the first time with ESI, prenols and mycolic acids (MAs). MAs are targets in antimycobacterial therapy, and they play an important immunomodulatory role during host-pathogen interactions. We compared high-resolution mass spectra of MAs derived from Mycobacterium bovis Bacille Camette-Guérin during entry into nonreplicative conditions induced by oxygen deprivation (hypoxic dormancy). Although the overall composition is not drastically altered, there are pronounced differences in individual MAs. alpha-MAs accumulate during entry into dormancy, whereas a subpopulation of keto-MAs is almost entirely eliminated. This effect is reversed upon resuscitation of dormant mycobacteria. These results provide detailed chemical information with relevance to drug development and immunobiology of mycobacteria.     

Disruption of tumor suppressor gene Hint1 leads to remodeling of the lipid metabolic phenotype of mouse liver

A lipidomic and metabolomic investigation of serum and liver from mice was performed to gain insight into the tumor suppressor gene Hint1. A major reprogramming of lipid homeostasis was found in both serum and liver of Hint1-null (Hint^−/−) mice, with significant changes in the levels of many lipid molecules, as compared with gender-, age-, and strain-matched WT mice. In the Hint1^−/− mice, serum total and esterified cholesterol were reduced 2.5-fold, and lysophosphatidylcholines (LPCs) and lysophosphatidic acids were 10-fold elevated in serum, with a corresponding fall in phosphatidylcholines (PCs). In the liver, MUFAs and PUFAs, including arachidonic acid (AA) and its metabolic precursors, were also raised, as was mRNA encoding enzymes involved in AA de novo synthesis. There was also a significant 50% increase in hepatic macrophages in the Hint1^−/− mice. Several hepatic ceramides and acylcarnitines were decreased in the livers of Hint1^−/− mice. The changes in serum LPCs and PCs were neither related to hepatic phospholipase A2 activity nor to mRNAs encoding lysophosphatidylcholine acetyltransferases 1-4. The lipidomic phenotype of the Hint1^−/− mouse revealed decreased inflammatory eicosanoids with elevated proliferative mediators that, combined with decreased ceramide apoptosis signaling molecules, may contribute to the tumor suppressor activity of Hint1.     

LC-MS/MS-based metabolic profiling of Escherichia coli under heterologous gene expression stress

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l -carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.     

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.     

Plasma phospholipid metabolic profiling and biomarkers of mouse IgA nephropathy

IgA nephropathy is the most common form of glomerulonephritis (GN) and it could progress to end-stage renal failure within 10 years. Participating in biological processes in various pathways, phospholipids as a class of important constituents in the biomembranes have been paid increasing attention in many fields. However, phospholipids metabolism in glomerular disease was not clear, especially in IgA nephropathy. In this paper, the plasma phospholipid metabolic profile in mouse IgA nephropathy was investigated to discover the potential biomarkers on the progression of this disease by using high performance liquid chromatography/mass spectrometry (HPLC/MS) and the principal components analysis (PCA) as well as partial least squares-discriminant analysis (PLS-DA). The experimental mouse models of IgA nephropathy were established by oral immune and BSA injection. It was found that expression of intercellular adhesion molecule-1 (ICAM-1) in the glomeruli had a significant correlation with proteinuria in mouse IgA nephropathy. The association between plasma phospholipids and expression of ICAM-1 in the glomeruli of IgA nephropathy suggested C18:0/C18:0 PS (phosphatidylserine), C18:0/C22:5 PS (phosphatidylserine) and C18:0/C20:4 PI (phosphatidylinositol) were possible biomarkers of IgA nephropathy. The results show that the plasma phospholipid metabolic profiles from HPLC/MS combining with PCA and PLS-DA can be used not only to differentiate the IgA nephropathy from the controls, but also to discover and identify the potential biomarkers.     

Plasma acylcarnitines and gut-derived aromatic amino acids as sex-specific hub metabolites of the human aging metabolome

Aging biology entails a cell/tissue deregulated metabolism that affects all levels of biological organization. Therefore, the application of “omic” techniques that are closer to phenotype, such as metabolomics, to the study of the aging process should be a turning point in the definition of cellular processes involved. The main objective of the present study was to describe the changes in plasma metabolome associated with biological aging and the role of sex in the metabolic regulation during aging. A high-throughput untargeted metabolomic analysis was applied in plasma samples to detect hub metabolites and biomarkers of aging incorporating a sex/gender perspective. A cohort of 1030 healthy human adults (45.9% females, and 54.1% males) from 50 to 98 years of age was used. Results were validated using two independent cohorts (1: n = 146, 53% females, 30–100 years old; 2: n = 68, 70% females, 19–107 years old). Metabolites related to lipid and aromatic amino acid (AAA) metabolisms arose as the main metabolic pathways affected by age, with a high influence of sex. Globally, we describe changes in bioenergetic pathways that point to a decrease in mitochondrial β-oxidation and an accumulation of unsaturated fatty acids and acylcarnitines that could be responsible for the increment of oxidative damage and inflammation characteristic of this physiological process. Furthermore, we describe for the first time the importance of gut-derived AAA catabolites in the aging process describing novel biomarkers that could contribute to better understand this physiological process but also age-related diseases.     

Review of Matrix-Assisted Laser Desorption Ionization-Imaging Mass Spectrometry for Lipid Biochemical Histopathology

Matrix-Assisted Laser Desorption Ionization-Imaging Mass Spectrometry (MALDI-IMS) is a rapidly evolving method used for the in situ visualization and localization of molecules such as drugs, lipids, peptides, and proteins in tissue sections. Therefore, molecules such as lipids, for which antibodies and other convenient detection reagents do not exist, can be detected, quantified, and correlated with histopathology and disease mechanisms. Furthermore, MALDI-IMS has the potential to enhance our understanding of disease pathogenesis through the use of “biochemical histopathology”. Herein, we review the underlying concepts, basic methods, and practical applications of MALDI-IMS, including post-processing steps such as data analysis and identification of molecules. The potential utility of MALDI-IMS as a companion diagnostic aid for lipid-related pathological states is discussed.     

Partial synthesis of ganglioside and lysoganglioside lipoforms as internal standards for MS quantification

Within recent years, ganglioside patterns have been increasingly analyzed by MS. However, internal standards for calibration are only available for gangliosides GM1, GM2, and GM3. For this reason, we prepared homologous internal standards bearing nonnatural fatty acids of the major mammalian brain gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, and of the tumor-associated gangliosides GM2 and GD2. The fatty acid moieties were incorporated after selective chemical or enzymatic deacylation of bovine brain gangliosides. For modification of the sphingoid bases, we developed a new synthetic method based on olefin cross metathesis. This method was used for the preparation of a lyso-GM1 and a lyso-GM2 standard. The total yield of this method was 8.7% for the synthesis of d17:1-lyso-GM1 from d20:1/18:0-GM1 in four steps. The title compounds are currently used as calibration substances for MS quantification and are also suitable for functional studies.     

Generation of a tightly regulated doxycycline-inducible model for studying mouse intestinal biology

To develop a sensitive and inducible system to study intestinal biology, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the 12.4 kb murine Villin promoter. The newly generated Villin-rtTA2-M2 mice were then bred with the previously developed tetO-HIST1H2BJ/GFP model to assess inducibility and tissue-specificity. Expression of the histone H2B-GFP fusion protein was observed exclusively upon doxycycline induction and was uniformly distributed throughout the intestinal epithelium. The Villin-rtTA2-M2 was also found to drive transgene expression in the developing mouse intestine. Furthermore, we could detect transgene expression in the proximal tubules of the kidney and in a population of alleged gastric progenitor cells. By administering different concentrations of doxycycline, we show that the Villin-rtTA2-M2 system drives transgene expression in a dosage-dependent fashion. Thus, we have generated a novel doxycycline-inducible mouse model, providing a valuable tool to study the effect of different gene dosages on intestinal physiology and pathology.