Identification of N-(2-propenal)ethanolamine as a urinary metabolite of malondialdehyde

N-(2-propenal)ethanolamine was isolated from rat and human urine using anion exchange, cation exchange, size exclusion and high performance liquid chromatography. Acid hydrolysis of the isolate yielded malondialdehyde (MDA) and ethanolamine (E) in a 1:1 molar ratio. A 1:1 E-MDA adduct was synthesized and found to be chromatographically inseparable from the urinary metabolite. Its NMR and UV spectra and lack of fluorescence were consistent with those of an enaminal formed by a Schiff's base reaction. The identification in urine of an adduct of MDA with ethanolamine, and the previous identification of an adduct with serine, constitutes direct evidence for the oxidative decomposition in vivo of polyunsaturated fatty acids present in the relevant phospholipids. The absence in urine of MDA adducts with other alpha-amino compounds (at least in comparable amounts) indicates that the ethanolamine and serine derivatives are formed in situ and not as a result of reactions with MDA generated in enzymatic processes.     

Identification of N-(2-propenal) serine as a urinary metabolite of malondialdehyde

N-2-(Propenal) serine (S-MDA) was synthesized by reacting serine with malondialdehyde (MDA) and was shown to be a 1:1 adduct of the starting materials. The synthetic compound was found to be identical to a metabolite of MDA excreted in rat and human urine. The identity of the metabolite was confirmed by isolation and hydrolysis to yield equimolar quantities of serine and MDA. The presence of S-MDA in urine constitutes direct evidence for oxidative decomposition of phospholipids by lipid peroxidation in vivo.     

Identification by proton NMR of N-(hydroxymethyl)-N-methylformamide as the major urinary metabolite of N,N-dimethylformamide in mice

Urine samples from mice which had received N,N-dimethylformamide were investigated by high field 1H-NMR spectroscopy. The most prominent signals in the N-CH3 region had chemical shifts identical with those of N,N-dimethylformamide (delta 2.85, 3.01) and N-(hydroxymethyl)-N-methylformamide (delta 2.91, 3.05). Resonances downfield of delta 7.5 (from formyl protons) also coincided with those of the reference formamides. When [14C]methyl-labelled N,N-dimethylformamide was injected and urine samples investigated by radio thin layer chromatography, the major area of radioactivity corresponded to the Rf of N-(hydroxymethyl)-N-methylformamide. Dimethylamine and methylamine were found to be minor metabolites of N,N-dimethylformamide.     

High-performance liquid chromatographic determination of N-epsilon-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate.

A recently reported methodology for amino acid analysis by HPLC has been adapted for quantification of N-epsilon-(2-propenal)lysine (a modified lysine by reaction with malondialdehyde that has been found in enzymatic digests of foods and in urine) in biological samples. We describe its use for investigating the in vitro degradation of N-epsilon-(2-propenal)lysine using rat tissue homogenates. Lysine dipeptide, used as a control in the incubation mixtures, and the lysine released by the hydrolytic action of the homogenates in the in vitro incubations are quantified in the same way. The samples are subjected to a cleanup prederivatization step using PD-10 disposable columns (Pharmacia). This allows precolumn derivatization with diethylethoxymethylenemalonate (50 min, 50 degrees C) and resolution of the derivatives of the compounds of interest by reversed-phase HPLC (binary gradient, 45 min) with quantification based on the uv absorption of the derivatives at 280 nm (detection limits below 1 pmol). The entire analysis takes 110 min. This method can be of general use for the determination of N-epsilon-(2-propenal)lysine in the context of research dealing with protein deterioration by reaction with malondialdehyde in biological systems and in foods. A method for the synthesis of N-epsilon-(2-propenal)lysine, used as external standard for the HPLC analysis, is described.     

Identification of osimertinib (AZD9291) as a lysine specific demethylase 1 inhibitor

Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that has been approved for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC). In this study, osimertinib was characterized as a LSD1 inhibitor for the first time with an IC₅₀ of 3.98 ± 0.3 μM and showed LSD1 inhibitory effect at cellular level. These findings provide new molecular skeleton for dual inhibitor for LSD1 and EGFR. Osimertinib could serve as a lead compound for further development for anti-NSCLC drug discovery with dual targeting.     

Aging increases Nepsilon-(carboxymethyl)lysine and caloric restriction decreases Nepsilon-(carboxyethyl)lysine and Nepsilon-(malondialdehyde)lysine in rat heart mitochondrial proteins

The present investigation studies the effect of aging, short-term and long-term caloric restriction on four different markers of oxidative, glycoxidative or lipoxidative damage to heart mitochondrial proteins: protein carbonyls (measured by ELISA); N epsilon -(carboxyethyl)lysine (CEL), N epsilon -(carboxymethyl)lysine (CML), and N epsilon -(malondialdehyde)lysine (MDA-lys) measured by gas chromatography/mass spectrometry. Aging increased the steady state level of CML in rat heart mitochondria without changing the levels of the other three markers of protein damage. Short-term caloric restriction (six weeks) did not change any of the parameters measured. However, long-term (one year) caloric restriction decreased CEL and MDA-lys in heart mitochondria and did not change protein carbonyls and CML levels. The decrease in MDA-lys was not due to changes in the sensitivity of mitochondrial lipids to peroxidation since the measurements of the fatty acid composition showed that the total number of fatty acid double bonds was not changed by caloric restriction. The decrease in CEL and MDA-lys in caloric restriction agrees with the previously and consistently described finding that caloric restriction agrees with the previously and consistently described finding that caloric restriction lowers the rate of generation of reactive oxygen species (ROS) in rodent heart mitochondria, although in the case of CEL a caloric restriction-induced lowering of glycaemia can also be involved. The CEL and MDA-lys results support the notion that caloric restriction decreases oxidative stress-derived damage to heart mitochondrial proteins.     

Identification and quantification of 6 beta-hydroxydexamethasone as a major urinary metabolite of dexamethasone in man

Identification of 6 beta-hydroxydexamethasone as a major urinary metabolite of dexamethasone in man has been accomplished by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Mass fragmentographic measurements revealed that more than 30% of the intravenously or orally administered dexamethasone dose was excreted in the 24-h urine as 6 beta-hydroxydexamethasone, while only a small fraction of the dose was excreted as unchanged dexamethasone and its glucuronic acid conjugate.     

Aging Increases N epsilon -(Carboxymethyl)lysine and Caloric Restriction Decreases N epsilon -(Carboxyethyl)lysine and N epsilon -(Malondialdehyde)lysine in Rat Heart Mitochondrial Proteins

The present investigation studies the effect of aging, short-term and long-term caloric restriction on four different markers of oxidative, glycoxidative or lipoxidative damage to heart mitochondrial proteins: protein carbonyls (measured by ELISA); N epsilon -(carboxyethyl)lysine (CEL), N epsilon -(carboxymethyl)lysine (CML), and N epsilon -(malondialdehyde)lysine (MDA-lys) measured by gas chromatography/mass spectrometry. Aging increased the steady state level of CML in rat heart mitochondria without changing the levels of the other three markers of protein damage. Short-term caloric restriction (six weeks) did not change any of the parameters measured. However, long-term (one year) caloric restriction decreased CEL and MDA-lys in heart mitochondria and did not change protein carbonyls and CML levels. The decrease in MDA-lys was not due to changes in the sensitivity of mitochondrial lipids to peroxidation since the measurements of the fatty acid composition showed that the total number of fatty acid double bonds was not changed by caloric restriction. The decrease in CEL and MDA-lys in caloric restriction agrees with the previously and consistently described finding that caloric restriction agrees with the previously and consistently described finding that caloric restriction lowers the rate of generation of reactive oxygen species (ROS) in rodent heart mitochondria, although in the case of CEL a caloric restriction-induced lowering of glycaemia can also be involved. The CEL and MDA-lys results support the notion that caloric restriction decreases oxidative stress-derived damage to heart mitochondrial proteins.     

Identification of lysine succinylation as a new post-translational modification

Of the 20 ribosomally coded amino acid residues, lysine is the most frequently post-translationally modified, which has important functional and regulatory consequences. Here we report the identification and verification of a previously unreported form of protein post-translational modification (PTM): lysine succinylation. The succinyllysine residue was initially identified by mass spectrometry and protein sequence alignment. The identified succinyllysine peptides derived from in vivo proteins were verified by western blot analysis, in vivo labeling with isotopic succinate, MS/MS and HPLC coelution of their synthetic counterparts. We further show that lysine succinylation is evolutionarily conserved and that this PTM responds to different physiological conditions. Our study also implies that succinyl-CoA might be a cofactor for lysine succinylation. Given the apparent high abundance of lysine succinylation and the significant structural changes induced by this PTM, it is expected that lysine succinylation has important cellular functions.     

Identification of the major urinary metabolite of the highly reactive cyclopentenone isoprostane 15-A(2t)-isoprostane in vivo

The cyclopentenone isoprostanes (A(2)/J(2)-IsoPs) are formed in significant amounts in humans and rodents esterified in tissue phospholipids. Nonetheless, they have not been detected unesterified in the free form, presumably because of their marked reactivity. A(2)/J(2)-IsoPs, similar to other electrophilic lipids such as 15-deoxy-Delta(12,14)-prostaglandin J(2) and 4-hydroxynonenal, contain a highly reactive alpha,beta-unsaturated carbonyl, which allows these compounds to react with thiol-containing biomolecules to produce a range of biological effects. We sought to identify and characterize in rats the major urinary metabolite of 15-A(2t)-IsoP, one of the most abundant A(2)-IsoPs produced in vivo, in order to develop a specific biomarker that can be used to quantify the in vivo production of these molecules. Following intravenous administration of 15-A(2t)-IsoP containing small amounts of [(3)H(4)]15-A(2t)-IsoP, 80% of the radioactivity excreted in the urine remained in aqueous solution after extraction with organic solvents, indicating the formation of a polar conjugate(s). Using high pressure liquid chromatography/mass spectrometry, the major urinary metabolite of 15-A(2t)-IsoP was determined to be the mercapturic acid sulfoxide conjugate in which the carbonyl at C9 was reduced to an alcohol. The structure was confirmed by direct comparison to a synthesized standard and via various chemical derivatizations. In addition, this metabolite was found to be formed in significant quantities in urine from rats exposed to an oxidant stress. The identification of this metabolite combined with the finding that these metabolites are produced in in vivo settings of oxidant stress makes it possible to use this method to quantify, for the first time, the in vivo production of cyclopentenone prostanoids.     

Immunochemical detection of a lipofuscin-like fluorophore derived from malondialdehyde and lysine

The accumulation of fluorescent age pigment or lipofuscin is a frequently observed age-associated cellular alteration in a variety of postmitotic cells of many species. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. In the present study, we raised a monoclonal antibody (MAb 1F83) directed to the malondialdehyde-modified protein and identified a lipofuscin-like dihydropyridine fluorophore as the major epitope. This antibody was used to conclusively demonstrate that the fluorophore forms on oxidatively modified low density lipoproteins. In addition, we demonstrated that the materials immunoreactive to MAb 1F83 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells. The results of this study suggest that the reaction between the lipid peroxidation-derived aldehyde and primary amino groups of protein might represent a process common to the formation of the lipofuscin-like fluorophore during aging and its related diseases.     

Identification of the major urinary metabolite of thromboxane B2 in the monkey.

Thromboxane B2 (TxB2) was biosynthesized from prostaglandin endoperoxides (PGG2, PGH2) using guinea pig lung microsomes and infused into an unanesthetized monkey. Urine was collected and TxB2 metabolites were isolated by reversed phase partition chromatography and high performance liquid chromatography. A major metabolite (TxB2-M) was found to be excreted in greater than two-fold abundance relative to other metabolites. Its structure was determined by gas chromatography-mass spectrometry to be dinor-thromboxane B2. In vitro incubation of TxB2 with rat liver mitochondria yielded a C18 derivative with a mass spectrum identical to that of TxB2-M, substantiating that the major urinary metabolite of TxB2 in the monkey is a product of a single step of beta-oxidation.     

Identification of lysine 37 of histone H2B as a novel site of methylation

Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined biological outcomes mediated by effector proteins that recognize such marks. While significant progress has been made in the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require a complete understanding of all the modifications that putatively contribute to it. Here, using the top-down mass spectrometry approach for identifying PTMs on full-length histones, we report that lysine 37 of histone H2B is dimethylated in the budding yeast Saccharomyces cerevisiae. By generating a modification-specific antibody and yeast strains that harbor mutations in the putative site of methylation, we provide evidence that this mark exist in vivo. Importantly, we show that this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in higher eukaryotes. By identifying a novel site of histone methylation, this study adds to our overall understanding of the complex number of histone modifications that contribute to chromatin function.     

Liquid chromatography-based determination of urinary free and total N(epsilon)-(carboxymethyl)lysine excretion in normal and diabetic subjects

We propose a specific, reproducible and sensitive HPLC method for the determination of N(epsilon)-(carboxymethyl)lysine (CML) excreted in urine. Total CML was measured in acid hydrolysates of urine samples, while free CML was measured in acetonitrile-deproteinised urine samples using a RP-HPLC method with ortho-phtaldialdehyde (OPA)-derivatisation and fluorescence detection suited for automation. We compared the CML excretion of 51 non-proteinuric patients with diabetes mellitus (DM) (age 57+/-14 years, HbA1c 8.0+/-1.8%) to 42 non-diabetic controls (C) (age 45+/-17 years). The urinary excretion of total CML in diabetic patients was increased by approximately 30% (DM: 0.58+/-0.21; C: 0.45+/-0.14 microM/mmol creatinine; P<0.001). While urinary excretion of free CML was not significantly different, excretion of bound CML was increased (DM: 0.36+/-0.17; C: 0.27+/-0.14; P<0.05) in diabetic patients. CML excretion was correlated with protein and albumin excretion, but did not correlate with HbA1c, duration of DM or diabetic complications such as neuropathy or retinopathy. Furthermore, no age-dependent change of total CML excretion was found, while free CML excretion was lower in younger subjects. The specific and sensitive determination of CML by RP-HPLC of its OPA-derivative is well suited for automation and better than that of less defined glycoxidation products (AGEs).     

Identification of S-1,2,2-trichlorovinyl-N-acetylcysteine as a urinary metabolite of tetrachloroethylene: bioactivation through glutathione conjugation as a possible explanation of its nephrocarcinogenicity

The elimination and metabolism of [14-C]-tetrachloroethylene (Tetra) was studied in female rats and mice after the oral administration of 800 mg/kg [14-C]-Tetra. Elimination of unchanged Tetra was the main pathway of elimination in both species and amounted to 91.2% of the dose in rats and 85.1% in mice. [14-C]-Carbon dioxide (CO2) was found to be a trace metabolite of [14-C]-Tetra. Only a small part of the applied dose was transformed to urinary (rats = 2.3%, mice = 7.1%) and fecal (rats = 2.0%, mice = 0.5%) metabolites. The urinary metabolites were separated and quantified by high performance liquid chromatography (HPLC) and identified by gas liquid chromatography/mass spectrometry (GC/MS). The following metabolites could be identified: oxalic acid (8.0% of urinary radioactivity in rats, 2.9% in mice), dichloroacetic acid (5.1%, 4.4%), trichloroacetic acid (54.0%, 57.8%), N-trichloroacetyl-aminoethanol (5.4%, 5.7%), trichloroethanol, free and conjugated (8.7%, 8.0%), S-1,2,2-trichlorovinyl-N-acetylcysteine (N-acetyl TCVC) (1.6%, 0.5%), and another conjugate of trichloroacetic acid (1.8%, 1.3%). The structures of the identified metabolites indicate two different pathways operative in Tetra biotransformation: cytochrome P-450-mediated epoxidation forming reactive metabolites in the liver and conjugation of Tetra with glutathione (GSH) catalyzed by glutathione transferase(s). The formation of reactive intermediates by renal processing of the glutathione conjugates may provide a molecular mechanism for the nephrotoxicity and nephrocarcinogenicity of Tetra in male rats.     

Identification of iso-TC-1 as a new T-2 toxin metabolite in cow urine

The structure of a new metabolite T-2 toxin (iso-TC-1) has been established as 3,15-diacetoxy-4-hydroxy-8(3-methyl-3'-hydroxy-butyryloxy)-12, 13-epoxytrichothec-9-ene. The compound is an isomer of TC-1 (a recently isolated T-2 derivative) in which the hydroxy and acetoxy groups at the C-3 and C-4 positions, respectively, are reversed. Direct probe analysis by electron impact (EI) of the underivatized iso-TC-1, as well as EI, positive chemical ionization (CI) in methane, and positive CI in ammonia of its trimethylsilylether or trifluoroacetate provided evidence to support the structure assignment of the new metabolite. The mass spectra of iso-TC-1 were compared with those of TC-1, T-2 toxin and iso-T-2 toxin (the isomer of T-2 toxin having reversed substituents at C-3 and C-4) with regard to molecular weight and fragments involving the substituents at C-3, C-4, C-8 and C-15. Although the two isomers, TC-1 and iso-TC-1, were not easily resolved by thin layer chromatography (TLC), a very good separation of their trimethylsilyl and trifluoroacetate derivatives was obtained by capillary gas chromatography. Acetylation of TC-1 or iso-TC-1 gave the same product. Iso-TC-1 is one of the main products of T-2 metabolism in the cow (more abundant than TC-1) and is found in the urine.     

Identification of dichloromethyl carbene as a metabolite of carbon tetrachloride

Although indirect evidence has suggested that liver microsomal cytochrome P-450 can reductively dehalogenate several compounds to carbene metabolites, there has been no direct proof for the formation of these reactive species. We report in this paper that carbenes can be chemically trapped and identified as metabolites. For example, 1,1-dichloro-2,2,3,3-tetramethylcyclopropane was identified as a metabolite by gas chromatography mass spectrometry when carbon tetrachloride (CCl₄) was incubated anaerobically with rat liver microsomes, NADPH and 2,3-dimethyl-2-butene. The reaction required NADPH and was inhibited by carbon monoxide. These findings show that cytochrome P-450 in rat liver microsomes can reductively metabolize CCl₄ to dichloromethyl carbene (:CCl₂) which can be trapped with 2,3-dimethyl-2-butene to form 1,1-dichloro-2,2,3,3-tetramethylcyclopropane. A similar approach may be used for the identification of carbene metabolites of other compounds.