Elevated lipoxygenase and cytochrome P450 products predict progression of chronic kidney disease.

BACKGROUND: The clinical relevance of arachidonic acid (AA) metabolites in chronic kidney disease (CKD) progression is poorly understood. We aimed to compare the concentrations of 85 enzymatic pathway products of AA metabolism in patients with CKD who progressed to end-stage kidney disease (ESKD) versus patients who did not in a subcohort of Chronic Renal Insufficiency Cohort (CRIC) and to estimate the risk of CKD progression and major cardiovascular events by levels of AA metabolites and their link to enzymatic metabolic pathways. METHODS: A total 123 patients in the CRIC study who progressed to ESKD were frequency matched with 177 nonprogressors and serum eicosanoids were quantified by mass spectrometry. We applied serum collected at patients' Year 1 visit and outcome of progression to ESKD was ascertained over the next 10 years. We used logistic regression models for risk estimation. RESULTS: Baseline 15-hydroxyeicosatetraenoate (HETE) and 20-HETE levels were significantly elevated in progressors (false discovery rate Q ≤ 0.026). The median 20-HETE level was 7.6 pmol/mL [interquartile range (IQR) 4.2-14.5] in progressors and 5.4 pmol/mL (IQR 2.8-9.4) in nonprogressors (P < 0.001). In an adjusted model, only 20-HETE independently predicted CKD progression. Each 1 standard deviation increase in 20-HETE was independently associated with 1.45-fold higher odds of progression (95% confidence interval 1.07-1.95; P = 0.017). Principal components of lipoxygenase (LOX) and cytochrome P450 (CYP450) pathways were independently associated with CKD progression. CONCLUSIONS: We found higher odds of CKD progression associated with higher 20-HETE, LOX and CYP450 metabolic pathways. These alterations precede CKD progression and may serve as targets for interventions aimed at halting progression.

Proposing a validation scheme for 13C metabolite tracer studies in high-resolution mass spectrometry.

13C metabolite tracer and metabolic flux analyses require upfront experimental planning and validation tools. Here, we present a validation scheme including a comparison of different LC methods that allow for customization of analytical strategies for tracer studies with regard to the targeted metabolites. As the measurement of significant changes in labeling patterns depends on the spectral accuracy, we investigate this aspect comprehensively for high-resolution orbitrap mass spectrometry combined with reversed-phase chromatography, hydrophilic interaction liquid chromatography, or anion-exchange chromatography. Moreover, we propose a quality control protocol based on (1) a metabolite containing selenium to assess the instrument performance and on (2) in vivo synthesized isotopically enriched Pichia pastoris to validate the accuracy of carbon isotopologue distributions (CIDs), in this case considering each isotopologue of a targeted metabolite panel. Finally, validation involved a thorough assessment of procedural blanks and matrix interferences. We compared the analytical figures of merit regarding CID determination for over 40 metabolites between the three methods. Excellent precisions of less than 1% and trueness bias as small as 0.01-1% were found for the majority of compounds, whereas the CID determination of a small fraction was affected by contaminants. For most compounds, changes of labeling pattern as low as 1% could be measured. Graphical abstract.

Application of differential mobility-mass spectrometry for untargeted human plasma metabolomic analysis.

Differential mobility spectrometry (DMS) has been gaining popularity in small molecule analysis over the last few years due to its selectivity towards a variety of isomeric compounds. While DMS has been utilized in targeted liquid chromatography-mass spectrometry (LC-MS), its use in untargeted discovery workflows has not been systematically explored. In this contribution, we propose a novel workflow for untargeted metabolomics based solely on DMS separation in a clinically relevant chronic kidney disease (CKD) patient population. We analyzed ten plasma samples from early- and late-stage CKD patients. Peak finding, alignment, and filtering steps performed on the DMS-MS data yielded a list of 881 metabolic features (unique mass-to-charge and migration time combinations). Differential analysis by CKD patient group revealed three main features of interest. One of them was putatively identified as bilirubin based on high-accuracy MS data and comparison of its optimum compensation voltage (COV) with that of an authentic standard. The DMS-MS analysis was four times faster than a typical HPLC-MS run, which suggests a potential for the utilization of this technique in screening studies. However, its lower separation efficiency and reduced signal intensity make it less suitable for low-abundant features. Fewer features were detected by the DMS-based platform compared with an HPLC-MS-based approach, but importantly, the two approaches resulted in different features. This indicates a high degree of orthogonality between HPLC- and DMS-based approaches and demonstrates the need for larger studies comparing the two techniques. The workflow described here can be adapted for other areas of metabolomics and has a value as a prescreening method to develop semi-targeted workflows and as a faster alternative to HPLC in large biomedical studies.

Lipidomics and Biomarker Discovery in Kidney Disease.

Technological advances in mass spectrometry-based lipidomic platforms have provided the opportunity for comprehensive profiling of lipids in biological samples and shown alterations in the lipidome that occur in metabolic disorders. A lipidomic approach serves as a powerful tool for biomarker discovery and gaining insight to molecular mechanisms of disease, especially when integrated with other -omics platforms (ie, transcriptomics, proteomics, and metabolomics) in the context of systems biology. In this review, we describe the workflow commonly applied to the conduct of lipidomic studies including important aspects of study design, sample preparation, biomarker identification and quantification, and data processing and analysis, as well as crucial considerations in clinical applications. We also review some recent studies of the application of lipidomic platforms that highlight the potential of lipid biomarkers and add to our understanding of the molecular basis of kidney disease.

Probing the application range and selectivity of a differential mobility spectrometry-mass spectrometry platform for metabolomics.

Metabolomics applications of differential mobility spectrometry (DMS)-mass spectrometry (MS) have largely concentrated on targeted assays and the removal of isobaric or chemical interferences from the signals of a small number of analytes. In the work reported here, we systematically investigated the application range of a DMS-MS method for metabolomics using more than 800 authentic metabolite standards as the test set. The coverage achieved with the DMS-MS platform was comparable to that achieved with chromatographic methods. High orthogonality was observed between hydrophilic interaction liquid chromatography and the 2-propanol-mediated DMS separation, and previously observed similarities were confirmed for the DMS platform and reversed-phase liquid chromatography. We describe the chemical selectivity observed for selected subsets of the metabolite test set, such as lipids, amino acids, nucleotides, and organic acids. Furthermore, we rationalize the behavior and separation of isomeric aromatic acids, bile acids, and other metabolites. Graphical abstract Differential mobility spectrometry-mass spectrometry (DMS-MS) facilitates rapid separation of metabolites of similar mass-to-charge ratio by distributing them across the compensation voltage range on the basis of their different molecular structures.

Effect of different immobilization strategies on chiral recognition properties of Cinchona-based anion exchangers.

In the enantiomeric separation of highly polar compounds, a traditionally challenging task for high-performance liquid chromatography, ion-exchange chiral stationary phases have found the main field of application. In this contribution, we present a series of novel anion-exchange-type chiral stationary phases for enantiomer separation of protected amino phosphonates and N-protected amino acids. Two of the prepared selectors possessed a double and triple bond within a single molecule. Thus, they were immobilized onto silica support employing either a thiol-ene (radical) or an azide-yne (copper(I)-catalyzed) click reaction. We evaluated the selectivity and the effect of immobilization proceeding either by the double bond of the Cinchona alkaloid or a triple bond of the carbamoyl moiety on the chromatographic performance of the chiral stationary phases using analytes with protecting groups of different size, flexibility, and π-acidity. The previously observed preference toward protecting groups possessing π-acidic units, which is a typical feature of Cinchona-based chiral stationary phases, was preserved. In addition, increasing the bulkiness of the selectors' carbamoyl units leads to significantly reduced retention times, while very high selectivity toward the tested analytes is retained.

Impaired ß-Oxidation and Altered Complex Lipid Fatty Acid Partitioning with Advancing CKD.

Studies of lipids in CKD, including ESRD, have been limited to measures of conventional lipid profiles. We aimed to systematically identify 17 different lipid classes and associate the abundance thereof with alterations in acylcarnitines, a metric of ß-oxidation, across stages of CKD. From the Clinical Phenotyping Resource and Biobank Core (CPROBE) cohort of 1235 adults, we selected a panel of 214 participants: 36 with stage 1 or 2 CKD, 99 with stage 3 CKD, 61 with stage 4 CKD, and 18 with stage 5 CKD. Among participants, 110 were men (51.4%), 64 were black (29.9%), and 150 were white (70.1%), and the mean (SD) age was 60 (16) years old. We measured plasma lipids and acylcarnitines using liquid chromatography-mass spectrometry. Overall, we identified 330 different lipids across 17 different classes. Compared with earlier stages, stage 5 CKD associated with a higher abundance of saturated C16-C20 free fatty acids (FFAs) and long polyunsaturated complex lipids. Long-chain-to-intermediate-chain acylcarnitine ratio, a marker of efficiency of ß-oxidation, exhibited a graded decrease from stage 2 to 5 CKD (P<0.001). Additionally, multiple linear regression revealed that the long-chain-to-intermediate-chain acylcarnitine ratio inversely associated with polyunsaturated long complex lipid subclasses and the C16-C20 FFAs but directly associated with short complex lipids with fewer double bonds. We conclude that increased abundance of saturated C16-C20 FFAs coupled with impaired ß-oxidation of FFAs and inverse partitioning into complex lipids may be mechanisms underpinning lipid metabolism changes that typify advancing CKD.

Glioblastoma Therapy Can Be Augmented by Targeting IDH1-Mediated NADPH Biosynthesis.

NADPH is a critical reductant needed in cancer cells to fuel the biosynthesis of deoxynucleotides and antioxidants and to sustain stress-survival responses after radiation-induced DNA damage. Thus, one rational strategy to attack cancer cells is to target their heavy reliance on NADPH. Here, we report that the isocitrate dehydrogenase IDH1 is the most strongly upregulated NADPH-producing enzyme in glioblastoma (GBM). IDH1 silencing in GBM cells reduced levels of NADPH, deoxynucleotides, and glutathione and increased their sensitivity to radiation-induced senescence. Rescuing these metabolic restrictions was sufficient to reverse IDH1-mediated radiosensitization. In a murine xenograft model of human GBM, we found that IDH1 silencing significantly improved therapeutic responses to fractionated radiotherapy, when compared with either treatment alone. In summary, our work offers a mechanistic rationale for IDH1 inhibition as a metabolic strategy to improve the response of GBM to radiotherapy. Cancer Res; 77(4); 960-70. ©2016 AACR.

Evaluation of coverage, retention patterns, and selectivity of seven liquid chromatographic methods for metabolomics.

Liquid chromatography-mass spectrometry-based metabolomics studies require highly selective and efficient chromatographic techniques. Typically employed reversed-phase (RP) methods fail to target polar metabolites, but the introduction of hydrophilic interaction liquid chromatography (HILIC) is slow due to perceived issues of reproducibility and ruggedness and a limited understanding of the complex retention mechanisms. In this study, we present a comparison of the chromatographic performance of a traditional RP-C18 column with zwitterionic, amide-, alkyl diol-, and aminoalkyl-based HILIC and mixed-mode columns. Our metabolite library represents one of the largest analyte sets available and consists of 764 authentic metabolite standards, including amino acids, nucleotides, sugars, and other metabolites, representing all major biological pathways and commonly observed exogenous metabolites (drugs). The coverage, retention patterns, and selectivity of the individual methods are highly diverse even between conceptually related HILIC methods. Furthermore, we show that HILIC sorbents having highly orthogonal selectivity and specificity enhance the coverage of major metabolite groups in (semi-) targeted applications compared to RP. Finally, we discuss issues encountered in the analysis of biological samples based on the results obtained with human plasma extracts. Our results demonstrate that fast and highly reproducible separations on zwitterionic columns are feasible, but knowledge of analyte properties is essential to avoid chromatographic bias and exclusion of key analytes in metabolomics studies. Graphical Abstract The chromatographic parameters of 764 authentic metabolite standards provide the basis for a comparison of coverage, selectivity and orthogonality of 7 reversed-phase (RP), mixed-mode (MM) and hydrophilic interaction liquid chromatography (HILIC) methods.

Application of cinchona-sulfonate-based chiral zwitterionic ion exchangers for the separation of proline-containing dipeptide rotamers and determination of on-column isomerization parameters from dynamic elution profiles.

The interconversion of cis and trans isomers of dipeptides containing C-terminal proline was studied by dynamic chromatography on zwitterionic chiral stationary phases at temperatures ranging from -15°C to +45°C The cis-trans isomers could be separated below 0°C and above 0-10°C plateau formation and peak coalescence phenomena occurred, which is characteristic for a dynamic process at the time-scale of partitioning. At and above room temperature, full coalescence was observed, which allowed separations of enantiomers without interference from interconversion effects. Analysis of the dynamic elution profiles of the interconverting peptides allowed the determination of isomerization rate constants and thermodynamic activation parameters (isomerization enthalpy, entropy and activation energy). In accordance with established results, isomerization rates and thermodynamic parameters were found to depend on the nature of the N-terminal amino acid. Isomerization barriers were only slightly lower than values determined with other methods but significant differences in the relative contributions of the activation enthalpy and entropy as well as isomerization rates pointed toward selector-moderated isomerization dynamics.

2-Acyl-dimedones as UV-active protective agents for chiral amino acids: enantiomer separations of the derivatives on chiral anion exchangers.

2-Acetyldimedone and 12 related compounds were employed as UV-active pre-column derivatizing agents for amino acids. Direct enantioseparation of the products was achieved using chiral anion exchanger stationary phases in polar-organic mobile phase mode. Under basic conditions, the reagents´ cyclic ß-tricarbonyl motifs can give rise to exo- and endocyclic enols through tautomerization. However, with primary amines (proteinogenic and unusual amino acids, aminosulfonic and aminophosphonic acids), we exclusively observed the formation of exocyclic enamine-type products. Reaction yields depended strongly on the 2-acyl modification of the reagent; in particular, we observed a significant decrease when electronegative or sterically demanding substituents were present in α-position to the exocyclic carbonyl group. In addition to improving UV detectability of the products, the introduction of this protective group facilitated successful enantiomer separations of the amino acid derivatives on Cinchona-based chiral anion exchangers. Particularly high enantiomer selectivity was observed in combination with stationary phases bearing a new variation of selectors with π-acidic (electron-poor) bis(trifluoromethyl)phenyl groups. No racemization of the analytes occurred at any stage of the analytical method including the deprotection, which was achieved with hydrazine.

Diastereoselective discrimination of lysine-alanine-alanine peptides by zwitterionic cinchona alkaloid-based chiral selectors using electrospray ionization mass spectrometry.

Electrospray ionization-mass spectrometry (ESI-MS) was used to investigate stereoselective interactions between seven zwitterionic alkylsulfonate-modified cinchona alkaloid chiral selectors and biologically relevant lysine-alanine-alanine tripeptide and alanine-alanine dipeptide selectands in modified methanolic solutions. Ion intensities from full scan mass spectra were used to assess degrees of association, the ratios of which were used to calculate selectivities for different selector-selectand pairs. The results support prior work on similar systems using HPLC, in that binding is mediated in these systems primarily through the quinuclidine amine on the selector and the C-terminal carboxylate of the peptide. N(α)- and N(α), N(ɛ)-acetylated forms of the tripeptide were used to study the relative contribution to binding imparted by the presence of multiple basic amines on the tripeptide with the selectors; this was not previously investigated by HPLC. The ability of the sulfonate group on the selector to reach and preferentially interact with the N(ɛ)-amine on the side chain of lysine was revealed. Overall, in acidic methanol conditions (0.5% acetic acid), degrees of association ranged from 1.5% to 17%, and selectivities ranged from non-selective to a 5.5:1 preference for binding one peptide stereoisomer over another with a given chiral selector. In sodium acetate (100 µM)-modified methanol solutions, significant changes in degrees of association (ranging from 4% to 25%) and selectivities (ranging from non-selective to 4.2:1 preference) were observed. These mass spectrometry experiments help to clarify the chiral recognition mechanism for these selectors and suggest that retention and selectivity could be further modulated in HPLC experiments through the utilization of alkali salt-containing mobile phases.

Versatility of cinchona-based zwitterionic chiral stationary phases: enantiomer and diastereomer separations of non-protected oligopeptides utilizing a multi-modal chiral recognition mechanism.

Cinchona alkaloid-based zwitterionic chiral stationary phases with diverse sulfonic acid cation exchanger groups were applied for enantiomer separations of small homochiral oligopeptides (all-l/all-d Ala-n, Val-n, Phe-n) using slightly acidic polar-organic mobile phase conditions which facilitated the ionization of ion exchanger sites of the chiral selectors as well as promoting zwitterion formation of the chiral selectands. Our studies focused on retention factors and selectivity as the main chromatographic parameters illustrating selector-selectand interactions. The results confirmed that bulky, aromatic side chains such as the benzyl group of Phe are analyte-inherent structural features promoting enantiorecognition. Regarding the zwitterionic selectors, a flexible spacer between the anion exchanger-carbamoyl and the cation exchanger sites was found to enhance stereoselectivity toward bulky and aromatic peptides while a rigid cation exchanger moiety contributed favorably to enantiomer distinction of peptides with small, aliphatic side chains. The investigations included all 28 stereoisomers of Phe-2 through Phe-4 to demonstrate the applicability of zwitterionic chiral stationary phases for the separation of sequential stereoisomers. Selectivity was shown to be governed by the Cinchona alkaloid motif but heavily dependent on effective hydrogen bonding between the carbamoyl motif and the amide groups of the peptide backbone. Contrary to previous studies on enantioselectivity which found pseudo-enantiomeric behavior of quinine and quinidine-based CSPs, in peptide stereoisomer separations CSPs based on quinidine undeniably performed better.

Increments to chiral recognition facilitating enantiomer separations of chiral acids, bases, and ampholytes using Cinchona-based zwitterion exchanger chiral stationary phases.

The intramolecular distances of anion and cation exchanger sites of zwitterionic chiral stationary phases represent potential tuning sites for enantiomer selectivity. In this contribution, we investigate the influence of alkanesulfonic acid chain length and flexibility on enantiomer separations of chiral acids, bases, and amphoteric molecules for six Cinchona alkaloid-based chiral stationary phases in comparison with structurally related anion and cation exchangers. Employing polar-organic elution conditions, we observed an intramolecular counterion effect for acidic analytes which led to reduced retention times but did not impair enantiomer selectivities. Retention of amphoteric analytes is based on simultaneous double ion pairing of their charged functional groups with the acidic and basic sites of the zwitterionic selectors. A chiral center in the vicinity of the strong cation exchanger site is vital for chiral separations of bases. Sterically demanding side chains are beneficial for separations of free amino acids. Enantioseparations of free (un-derivatized) peptides were particularly successful in stationary phases with straight-chain alkanesulfonic acid sites, pointing to a beneficial influence of more flexible moieties. In addition, we observed pseudo-enantiomeric behavior of quinine and quinidine-derived chiral stationary phases facilitating reversal of elution orders for all analytes.