Sensitive and Reproducible Mass Spectrometry-Compatible RP-UHPLC Analysis of Tricarboxylic Acid Cycle and Related Metabolites in Biological Fluids: Application to Human Urine.

We describe a method for the analysis of organic acids, including those of the tricarboxylic acid cycle (TCA cycle), by mixed-mode reversed-phase chromatography, on a CSH Phenyl-Hexyl column, to accomplish mixed-mode anion-exchange separations, which results in increased retention for acids without the need for ion-pairing reagents or other mobile phase additives. The developed method exhibited good retention time reproducibility for over 650 injections or more than 5 days of continuous operation. Additionally, it showed excellent resolution of the critical pairs, isocitric acid and citric acid as well as malic acid and fumaric acid, among others. The use of hybrid organic-inorganic surface technology incorporated into the hardware of the column not only improved the mass spectral quality and subsequent database match scoring but also increased the recovery of the analytes, showing particular benefit for low concentrations of phosphorylated species. The method was applied to the comparative metabolomic analysis of urine samples from healthy controls and breast cancer positive subjects. Unsupervised PCA analysis showed distinct grouping of samples from healthy and diseased subjects, with excellent reproducibility of respective injection clusters. Finally, abundance plots of selected analytes from the tricarboxylic acid cycle revealed differences between healthy control and disease groups.

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

SIRT3 and GCN5L regulation of NADP+- and NADPH-driven reactions of mitochondrial isocitrate dehydrogenase IDH2.

Wild type mitochondrial isocitrate dehydrogenase (IDH2) was previously reported to produce oncometabolite 2-hydroxyglutarate (2HG). Besides, mitochondrial deacetylase SIRT3 has been shown to regulate the oxidative function of IDH2. However, regulation of 2HG formation by SIRT3-mediated deacetylation was not investigated yet. We aimed to study mitochondrial IDH2 function in response to acetylation and deacetylation, and focus specifically on 2HG production by IDH2. We used acetylation surrogate mutant of IDH2 K413Q and assayed enzyme kinetics of oxidative decarboxylation of isocitrate, 2HG production by the enzyme, and 2HG production in cells. The purified IDH2 K413Q exhibited lower oxidative reaction rates than IDH2 WT. 2HG production by IDH2 K413Q was largely diminished at the enzymatic and cellular level, and knockdown of SIRT3 also inhibited 2HG production by IDH2. Contrary, the expression of putative mitochondrial acetylase GCN5L likely does not target IDH2. Using mass spectroscopy, we further identified lysine residues within IDH2, which are the substrates of SIRT3. In summary, we demonstrate that 2HG levels arise from non-mutant IDH2 reductive function and decrease with increasing acetylation level. The newly identified lysine residues might apply in regulation of IDH2 function in response to metabolic perturbations occurring in cancer cells, such as glucose-free conditions.

A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1.

The enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the conversion of isocitrate to alpha-ketoglutarate (αKG) and has emerged as an important therapeutic target for glioblastoma multiforme (GBM). Current methods for assaying IDH1 remain poorly suited for high-throughput screening of IDH1 antagonists. This paper describes a high-throughput and quantitative assay for IDH1 that is based on the self-assembled monolayers for matrix-assisted laser desorption/ionization-mass spectrometry (SAMDI-MS) method. The assay uses a self-assembled monolayer presenting a hydrazide group that covalently captures the αKG product of IDH1, where it can then be detected by MALDI-TOF mass spectrometry. Co-capture of an isotopically-labeled αKG internal standard allows the αKG concentration to be quantitated. The assay was used to analyze a series of standard αKG solutions and produced minimal error in measured αKG concentration values. The suitability of the assay for high-throughput analysis was evaluated in a 384-sample biochemical IDH1 screen. Cells expressing IDH1 were lysed and the lysate was applied to the monolayer to capture αKG, which was then quantitated using the SAMDI-MS assay. Cells in which IDH1 expression was reduced by small-interfering RNA exhibited a corresponding decrease in αKG concentration as measured by the assay. Application of the assay toward the high-throughput screening of IDH1 inhibitors or knockdown agents may facilitate the discovery of treatments for GBM.

Amaranth's 2-Caffeoylisocitric Acid-An Anti-Inflammatory Caffeic Acid Derivative That Impairs NF-κB Signaling in LPS-Challenged RAW 264.7 Macrophages.

For centuries, Amaranthus sp. were used as food, ornamentals, and medication. Molecular mechanisms, explaining the health beneficial properties of amaranth, are not yet understood, but have been attributed to secondary metabolites, such as phenolic compounds. One of the most abundant phenolic compounds in amaranth leaves is 2-caffeoylisocitric acid (C-IA) and regarding food occurrence, C-IA is exclusively found in various amaranth species. In the present study, the anti-inflammatory activity of C-IA, chlorogenic acid, and caffeic acid in LPS-challenged macrophages (RAW 264.7) has been investigated and cellular contents of the caffeic acid derivatives (CADs) were quantified in the cells and media. The CADs were quantified in the cell lysates in nanomolar concentrations, indicating a cellular uptake. Treatment of LPS-challenged RAW 264.7 cells with 10 µM of CADs counteracted the LPS effects and led to significantly lower mRNA and protein levels of inducible nitric oxide synthase, tumor necrosis factor alpha, and interleukin 6, by directly decreasing the translocation of the nuclear factor κB/Rel-like containing protein 65 into the nucleus. This work provides new insights into the molecular mechanisms that attribute to amaranth's anti-inflammatory properties and highlights C-IA's potential as a health-beneficial compound for future research.

Mechanism-based inactivator of isocitrate lyases 1 and 2 from Mycobacterium tuberculosis.

Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator of Mtb ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys191 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalent S-homopyruvoyl adduct of the active-site Cys191.

Cryptoporic acid S, a new drimane-type sesquiterpene ether of isocitric acid from the fruiting bodies of Cryptoporus volvatus.

A new drimane-type sesquiterpene with an isocitric acid moiety, cryptoporic acid S (1), together with six known compounds, cryptoporic acid D (2), ß-sitosterol (3), ß-daucosterol (4), stigmast-4-en-3-one (5), ergosterol (6), and (22E,24R)-ergosta-7,22-diene-3ß,5α,6ß-triol (7), was isolated from the fruiting bodies of Cryptoporus volvatus. The structures of these compounds were established on the basis of UV, IR, MS, 1D and 2D NMR analysis. In the meanwhile, compounds 1 and 2 were evaluated for antioxidant activity using the methods of 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity (DPPH-RSA) and ferric reducing antioxidant power (FRAP) assay, and they exhibited moderate antioxidant activities.

Mutant IDH1 enhances the production of 2-hydroxyglutarate due to its kinetic mechanism.

The human, cytosolic enzyme isocitrate dehydrogenase 1 (IDH1) reversibly converts isocitrate to α-ketoglutarate (αKG). Cancer-associated somatic mutations in IDH1 result in a loss of this normal function but a gain in a new or neomorphic ability to convert αKG to the oncometabolite 2-hydroxyglutarate (2HG). To improve our understanding of the basis for this phenomenon, we have conducted a detailed kinetic study of wild-type IDH1 as well as the known 2HG-producing clinical R132H and G97D mutants and mechanistic Y139D and (newly described) G97N mutants. In the reductive direction of the normal reaction (αKG to isocitrate), dead-end inhibition studies suggest that wild-type IDH1 goes through a random sequential mechanism, similar to previous reports on related mammalian IDH enzymes. However, analogous experiments studying the reductive neomorphic reaction (αKG to 2HG) with the mutant forms of IDH1 are more consistent with an ordered sequential mechanism, with NADPH binding before αKG. This result was further confirmed by primary kinetic isotope effects for which saturating with αKG greatly reduced the observed isotope effect on (D)(V/K)NADPH. For the mutant IDH1 enzyme, the change in mechanism was consistently associated with reduced efficiencies in the use of αKG as a substrate and enhanced efficiencies using NADPH as a substrate. We propose that the sum of these kinetic changes allows the mutant IDH1 enzymes to reductively trap αKG directly into 2HG, rather than allowing it to react with carbon dioxide and form isocitrate, as occurs in the wild-type enzyme.

The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.

The somatic mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) observed in gliomas can lead to the production of 2-hydroxyglutarate (2HG). Here, we report that tumor 2HG is elevated in a high percentage of patients with cytogenetically normal acute myeloid leukemia (AML). Surprisingly, less than half of cases with elevated 2HG possessed IDH1 mutations. The remaining cases with elevated 2HG had mutations in IDH2, the mitochondrial homolog of IDH1. These data demonstrate that a shared feature of all cancer-associated IDH mutations is production of the oncometabolite 2HG. Furthermore, AML patients with IDH mutations display a significantly reduced number of other well characterized AML-associated mutations and/or associated chromosomal abnormalities, potentially implicating IDH mutation in a distinct mechanism of AML pathogenesis.

Down-regulation of iron regulatory protein 1 activities and expression in superoxide dismutase 1 knock-out mice is not associated with alterations in iron metabolism.

Iron and oxygen (O2) are intimately associated in many well characterized patho-physiological processes. These include oxidation of the [4Fe-4S] cluster of mitochondrial aconitase and inactivation of this Krebs cycle enzyme by the superoxide anion (O2*-), a product of the one-electron of reduction O2. In contrast to the apparent toxicity of this reaction, the biological consequences of O2*- -mediated inactivation of the cytosolic counterpart of mitochondrial aconitase, commonly known as iron regulatory protein 1 (IRP1), are not clear. Apart from its ability to convert citrate to iso-citrate, IRP1 in its apo-form binds to iron-responsive elements in the untranslated regions of mRNAs coding for proteins involved in iron metabolism, to regulate their synthesis and thus control the cellular homeostasis of this metal. Here, we show that in superoxide dismutase 1 (SOD1) knock-out mice, lacking Cu,Zn-SOD, an enzyme that acts to reduce the concentration of O2*- mainly in cytosol, not only is aconitase activity of IRP1 inhibited but the level of IRP1 is also strongly decreased. Despite such an evident alteration in IRP1 status, SOD1-deficient mice display a normal iron metabolism phenotype. Our findings clearly show that under conditions of O2*- -mediated oxidative stress, IRP1 is not essential for the maintenance of iron metabolism in mammals.

Isocitrate binding at two functionally distinct sites in yeast NAD+-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octamer containing two types of homologous subunits. Ligand-binding analyses were conducted to examine effects of residue changes in putative catalytic and regulatory isocitrate-binding sites respectively contained in IDH2 and IDH1 subunits. Replacement of homologous serine residues in either subunit site, S98A in IDH2 or S92A in IDH1, was found to reduce by half the total number of holoenzyme isocitrate-binding sites, confirming a correlation between detrimental effects on isocitrate binding and respective kinetic defects in catalysis and allosteric activation by AMP. Replacement of both serine residues eliminates isocitrate binding and measurable catalytic activity. The putative isocitrate-binding sites of IDH1 and IDH2 contain five identical and four nonidentical residues. Reciprocal replacement of the four nonidentical residues in either or both subunits (A108R, F136Y, T241D, and N245D in IDH1 and/or R114A, Y142F, D248T, and D252N in IDH2) was found to be permissive for isocitrate binding. This provides further evidence for two types of binding sites in IDH, although the authentic residues have been shown to be necessary for normal kinetic contributions. Finally, the mutant enzymes with residue replacements in the IDH1 site were found to be unable to bind AMP, suggesting that allosteric activation is dependent both upon binding of isocitrate at the IDH1 site and upon the changes in the enzyme normally elicited by this binding.

Affinity cleavage at the divalent metal site of porcine NAD-specific isocitrate dehydrogenase.

A divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD-dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The NAD-specific isocitrate dehydrogenase is composed of three distinct subunits in the ratio 2alpha:1beta:1gamma. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa alpha subunit of the enzyme. A pair of major peptides is generated during Fe2+ inactivation: 29.5 + 10.5 kDa, as determined by SDS-PAGE. Amino-terminal sequencing reveals that these peptides arise by cleavage of the Val262-His263 bond of the alpha subunit. No fragments are produced when enzyme is incubated with Fe2+ and ascorbate under denaturing conditions in the presence of 6 M urea, indicating that the native structure is required for the specific cleavage. These results suggest that His263 of the alpha subunit may be a ligand of the divalent metal ion needed for the reaction catalyzed by isocitrate dehydrogenase. Isocitrate enhances the inactivation of enzyme caused by Fe2+ in the presence of oxygen, but prevents the cleavage, suggesting that inactivation occurs by a different mechanism when metal ion is bound to the enzyme in the presence of isocitrate: oxidation of cysteine may be responsible for the rapid inactivation in this case. Affinity cleavage caused by Fe2+ implicates alpha as the catalytic subunit of the multisubunit porcine NAD-dependent isocitrate dehydrogenase.

The beta/alpha peak height ratio of ATP. A measure of free [Mg2+] using 31P NMR.

From 31P NMR measurements made in vitro at 38 degrees C, I = 0.25, pH 5. 75-8.5, and calculated free [Mg2+] from 0 to 5 mM, we show that, within the physiological range of cytosolic free [Mg2+] from 0.25 to 1.5 mM, the chemical shift difference between the alpha- and beta-ATP resonances, deltaalphabeta, changes by only 0.6 ppm. Consequently, we developed new formalisms from known acid and Mg2+ dissociation constants by which the observed chemical shift of Pi, deltaPi, and the peak height ratio of the beta- and alpha-ATP resonances, hbeta/alpha, could be related to free [Mg2+] by simultaneous solution of: [equation: see text] We found that hbeta/alpha changed 2.5-fold as free [Mg2+] varied from 0.25 to 1.5 mM, providing a more sensitive and accurate measure of free cytosolic [Mg2+]. In working rat heart perfused with glucose, free [Mg2+] was 1.0 +/- 0.1 from hbeta/alpha and 1.2 +/- 0.03 from measured [citrate]/[isocitrate] but 0.51 +/- 0.1 from deltaalphabeta. Addition of ketone bodies to the perfusate decreased free [Mg2+] estimated from hbeta/alpha to 0.61 +/- 0.02 and 0.74 +/- 0.11 by [citrate]/[isocitrate] but the estimate from deltaalphabeta was unchanged at 0.46 +/- 0.04 mM. Such differences in estimated free [Mg2+] alter the apparent Keq of the creatine kinase reaction and hence the estimated cytosolic free [SigmaADP].