Water soluble selenometabolome of Cardamine violifolia.

Low molecular weight selenium containing metabolites in the leaves of the selenium hyperaccumulator Cardamine violifolia (261 mg total Se per kg d.w.) were targeted in this study. One dimensional cation exchange chromatography coupled to ICP-MS was used for purification and fractionation purposes prior to LC-Unispray-QTOF-MS analysis. The search for selenium species in full scan spectra was assisted with an automated mass defect based filtering approach. Besides selenocystathionine, selenohomocystine and its polyselenide derivative, a total number of 35 water soluble selenium metabolites other than selenolanthionine were encountered, including 30 previously unreported compounds. High occurrence of selenium containing hexoses was observed, together with the first assignment of N-glycoside derivatives of selenolanthionine. Quantification of the most abundant selenium species, selenolanthionine, was carried out with an ion pairing LC - post column isotope dilution ICP-MS setup, which revealed that this selenoamino acid accounted for 30% of the total selenium content of the leaf (78 mg (as Se) per kg d.w.).

Urinary metabolomics revealed arsenic exposure related to metabolic alterations in general Chinese pregnant women.

Arsenic exposure is considered a major environmental threat to human health. It is already known that high-level arsenic exposure has adverse effects on human health. Since the pregnant women are known to be more susceptible to some chemical exposures than ordinary people, the understanding regarding the health effects of low-level arsenic exposure on pregnant women is critical and remains unclear. The aim of this study is to investigate the urinary metabolic changes of pregnant women exposed to low-dose arsenic, and to identify biomarkers from metabolomics analysis. Urine samples of 246 pregnant women were collected in the first trimester of pregnancy and were divided into three groups based on the tertile distribution of urinary arsenic concentrations which were determined using inductively coupled plasma mass spectrometry (ICP-MS). Changes in the metabolite profile were measured using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF MS). Arsenic- related metabolic biomarkers were investigated by comparing the samples of the first and third tertiles of arsenic exposure classifications using a partial least-squares discriminant model (PLS-DA). Nine urine potential biomarkers were putatively identified, including LysoPC (14:0), glutathione, 18-carboxy-dinor-LTE4, 20-COOH-LTE4, cystathionine ketimin, 1-(beta-d-ribofuranosyl)-1,4-dihydronicotinamide, thiocysteine, p-cresol glucuronide and vanillactic acid. The obtained results showed that environmental arsenic exposure, even at low levels, could cause metabolite alterations in pregnant women which might be associated with adverse health outcomes. This is the first report on metabolic changes in pregnant women for arsenic exposure. The findings may be valuable for the arsenic risk assessment for pregnant women.

A facile synthesis of differently protected cystathionines in aqueous solutions.

A simple method for the synthesis of differently protected cystathionines is described. Reaction of γ- bromohomoalanine with cysteine derivatives in an ethyl acetate/water two-phase system, which contains TBAHS and NaHCO3, led to the desired protected cystathionines in very high to excellent yields. Apart from the high yields, the mild conditions and the ease of conducting the reactions are also attractive features for this procedure.

Standardless identification of selenocystathionine and its gamma-glutamyl derivatives in monkeypot nuts by 3D liquid chromatography with ICP-MS detection followed by nanoHPLC-Q-TOF-MS/MS.

A three-step chromatographic procedure using orthogonal separation mechanisms (size-exclusion, cation-exchange and ion-pairing reversed phase) was developed to purify three low molecular weight selenospecies, including the major compound, from the aqueous extract of monkeypot (Lecythis minor) nuts. The following reversed-phase nanoHPLC-electrospray Q-TOF-MS/MS allowed the formal standardless identification of selenocystathionine and two isoforms of gamma-glutamyl-selenocystathionine. This is the first MS and MS/MS-based formal evidence of the presence of these compounds in a biological sample.

Yeast cystathionine beta-synthase reacts with L-allothreonine, a non-natural substrate, and L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine.

Our studies of the reaction mechanism of cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme. The enzyme catalyzes the reaction of L-serine with L-homocysteine to form L-cystathionine through a series of pyridoxal phosphate intermediates. In this work, we explore the substrate specificity of the enzyme by use of substrate analogues combined with kinetic measurements under pre-steady-state conditions and with circular dichroism and fluorescence spectroscopy under steady-state conditions. Our results show that L-allothreonine, but not L-threonine, serves as an effective substrate. L-Allothreonine reacts with the pyridoxal phosphate cofactor to form a stable 3-methyl aminoacrylate intermediate that absorbs maximally at 446 nm. The rapid-scanning stopped-flow results show that the binding of L-allothreonine as the external aldimine is faster than formation of the 3-methyl aminoacrylate intermediate. The 3-methyl aminoacrylate intermediate reacts with L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine, which was characterized by nuclear magnetic resonance spectroscopy. This new amino acid may be a useful analogue of L-cystathionine.

Accumulation of cystathionine, cystathionine ketimine, and perhydro-1,4-thiazepine-3,5-dicarboxylic acid in whole brain and various regions of the brain of D, L-propargylglycine-treated rats.

Experimental cystathioninuria was induced in rats by administration of the cystathionine gamma-lyase inhibitor, D,L-propargylglycine. The cystathionine metabolites, cystathionine ketimine (CK) and perhydro-1,4-thiazepine-3,5-dicarboxylic acid (PHTZDC), were identified in whole brain and various regions of the brain in D,L-propargylglycine-treated rats. The concentration of CK and PHTZDC in whole brain and various regions of the brain increased gradually after administration of D,L-propargylglycine, and reached the highest value at about 20 hours. CK and PHTZDC accumulated in whole brain and various regions of the brain in proportion to the amount of accumulated cystathionine after D,L-propargylglycine administration. The concentration of these compounds in the cerebellum was higher versus the other regions of the rat brain.

Novel priming compounds of cystathionine metabolites on superoxide generation in human neutrophils.

Human peripheral blood polymorphonuclear leukocytes were preincubated with cystathionine and cystathionine metabolites found in the urine of patients with cystathioninuria. Among the cystathionine metabolites, cystathionine ketimine and N-acetyl-S-(3-oxo-3-carboxy-n-propyl) cysteine (NAc-OCPC) significantly enhanced the N-formylmethionylleucylphenylalanine (fMLP)-induced superoxide generation, but cystathionine, NAc-cystathionine, and cyclothionine did not enhance the superoxide generation. Cystathionine ketimine and NAc-OCPC also enhanced superoxide generation induced by opsonized zymosan (OZ) but not that induced by arachidonic acid (AA) and phorbol 12-myristate 13-acetate (PMA). Superoxide generation induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein, an inhibitor of tyrosine kinase, and was enhanced by 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C. Cystathionine ketimine and NAc-OCPC markedly also increased phosphorylation of 45-kDa protein in human neutrophils and the phosphorylation depended on the concentrations of cystathionine ketimine and NAc-OCPC. The phosphorylation of 45-kDa protein induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein and herbimycin A, inhibitors of tyrosine kinase, but was not inhibited by H-7 and staurosporine, inhibitors of protein kinase C. Cystathionine metabolites and l-cystathionine sulfoxides were separated into two diastereoisomers, CS-I and CS-II. CS-I enhanced the superoxide generation induced by AA and PMA but not that induced by fMLP and OZ. In contrast, CS-II enhanced the superoxide generation induced by fMLP and OZ, but not that induced by AA and PMA.

D-cystathionine ketimine and L-cystathionine ketimine enhance superoxide generation by human neutrophils in a different manner.

The effects of d-cystathionine ketimine (D-CK) and l-cystathionine ketimine (L-CK) on the stimulus-induced superoxide generation by human neutrophils were compared. When the cells were preincubated with D-CK, the superoxide generation induced by arachidonic acid (AA), phorbol 12-myristate 13-acetate (PMA), and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were enhanced, showing a dependence on D-CK concentration. The rate of enhancement by D-CK was AA > PMA > fMLP. On the contrary, L-CK largely enhanced the fMLP-induced superoxide generation, whereas it showed no effect on those induced by AA and PMA. The superoxide generations induced by AA and PMA in the D-CK-treated cells were suppressed by staurosporine, while those in the L-CK-treated cells were not affected. Genistein suppressed the fMLP-induced superoxide generation in the L-CK-treated cells more efficiently than that in the D-CK-treated cells. D-CK enhanced seryl phosphorylation of 16. 5-kDa protein in human neutrophils, while L-CK enhanced tyrosyl phosphorylation of 45-kDa protein.

Metabolism of cystathionine, N-monoacetylcystathionine, perhydro-1,4-thiazepine-3,5-dicarboxylic acid, and cystathionine ketimine in the liver and kidney of D,L-propargylglycine-treated rats.

Experimental cystathioninuria was induced by injection of D,L-propargylglycine in rats. The novel cystathionine metabolites, N-monoacetylcystathionine (NAc-cysta), perhydro-1,4-thiazepine-3,5-dicarboxylic acid (PHTZDC), and cystathionine ketimine (CK), were identified previously in the urine of patients with cystathioninuria and D,L-propargylglycine-treated rats. In this study, we identified these compounds in the liver and kidney of D,L-propargylglycine-treated rats using liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system (LC/APCI-MS) and an amino acid analyzer. The metabolism of these compounds in the liver and kidney of D,L-propargylglycine-treated rats was also studied. PHTZDC, NAc-cysta, and CK were accumulated in the rat tissues in proportion to the content of cystathionine after D,L-propargylglycine administration. The concentrations of these compounds in the liver were higher than those in the kidney, and these compounds reached maxima earlier in the liver than in the kidney.

Different effect of diastereoisomers of L-cystathionine sulfoxide on the stimulus coupled responses of human neutrophils.

The priming effect of L-cystathionine sulfoxide, which is one of the unusual cystathionine metabolites found in the urine of patients with cystathioninuria, on the stimulus-induced superoxide generation by human neutrophils was examined. The synthetic L-cystathionine sulfoxide significantly enhanced the superoxide generations induced by N-formyl-methionyl-leucyl-phenylalanine [fMLP], opsonized zymosan [OZ], arachidonic acid [AA], and phorbol 12-myristate 13-acetate [PMA]. Then the synthetic L-cystathionine sulfoxide was separated into two diastereoisomers, CS-I and CS-II, which showed a peak at 76 and 83 min on chromatogram by amino acid analyzer, respectively. CS-I enhanced the superoxide generations induced by AA and PMA but not those induced by fMLP and OZ. On the contrary, CS-II enhanced the superoxide generations induced by fMLP and OZ but not those induced by AA and PMA. The superoxide generation induced by PMA with CS-I was suppressed by H-7 and was enhanced by genistein, while that by fMLP with CS-II was suppressed by genistein and was enhanced by H-7.

Detection of cystathionine ketimine and lanthionine ketimine in human brain.

The sulfur containing imino acids cystathionine ketimine (CK) and lanthionine ketimine (LK) have been detected in the human brain by an HPLC procedure. The HPLC procedure takes advantage of the selective absorbance at 380 nm of the phenylisothiocyanate-ketimine adduct. Quantitation of cystathionine ketimine and lanthionine ketimine indicates a mean concentration (mean +/- SD, n = 4) of 2.3 +/- 0.8 nmol/g for CK and of 1.1 +/- 0.3 nmol/g for LK in four human cerebral cortex samples of neurosurgical source. The identification of these cyclic ketimine derivatives of L-cystathionine and L-lanthionine as normal human metabolites in human nervous tissue may have interesting metabolic and physiological implications.

Identification of cyclic cystathionine sulfoxide and N-acetylcyclic cystathionine in the urine of a patient with cystathioninuria using liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system.

Perhydro-1,4-thiazepine-4,5-dicarboxylic acid sulfoxide (cyclic cystathionine sulfoxide [cyclic cystaSO]) and N-acetylperhydro-1,4-thiazepine-3,5-dicarboxylic acid (NAc-cyclic cysta) have been identified in the urine of a patient with cystathioninuria as new metabolites of cystathionine for the first time using liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system (LC/APCI-MS). The concentrations of cyclic cystaSO and NAc-cyclic cysta in the urine of a patient with cystathioninuria have also been determined for the first time using this method: 18.24 +/- 0.79 and 25.23 +/- 0.83 mg/g creatinine, respectively.

Effect of cystathionine ketimine on the stimulus coupled responses of neutrophils and their modulation by various protein kinase inhibitors.

Human peripheral blood polymorphonuclear leukocytes were perincubated with cystathionine and cystathionine metabolites found in the urine of the patients with cystathioninuria. Among the cystathionine metabolites, cystathionine ketimine significantly enhanced the N-formyl-methionyl-leucyl-phenylalanine-induced superoxide generation, but cystathionine and cyclothionine did not enhance the superoxide generation. Cystathionine ketimine also enhanced superoxide generation induced by opsonized zymosan but not those induced by arachidonic acid and phorbol myristate acetate. Superoxide generation induced by cystathionine ketimine was inhibited by genistein, an inhibitor of tyrosine kinase, and was enhanced by 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine, an inhibitor of protein kinase C.

On the oxidation of cystathionamine and selenocystathionamine by plant amineoxidase.

Cystathionamine and selenocystathionamine, diamines analogous to 1,6-diaminohexane but having the third methylene group of the carbon chain substituted by a S or a Se atom, are asymmetrical thio- (seleno-) ethers. They can give rise by oxidative monodeamination to two different aminoaldehydes. It has been shown that lentil seedlings amineoxidase catalyzes the oxidative deamination of either the one or the other aminogroup of cystathionamine or of selenocystathionamine, giving rise to both possible aminoaldehydes.

Identification of new cystathionine mono-oxo acids, S-(3-oxo-3-carboxy-n-propyl) cysteine and S-(2-oxo-2-carboxyethyl) homocysteine, in the urine of a patient with cystathioninuria.

Novel cystathionine mono-oxo acids, S-(3-oxo-3-carboxy-n-propyl) cysteine and S-(2-oxo-2-carboxyethyl) homocysteine, and cyclic amino acid, cystathionine ketimine, have been detected in the urine of a patient with cystathioninuria using liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface system and an amino acid analyzer. To determine these cystathionine mono-oxo acids and cystathionine ketimine we took advantage of the selective absorbance at 380 nm of the phenylisothiocyanate-ketimine interaction product. The total concentrations of these compounds found in the urine samples of a cystathioninuric patient and six healthy subjects were respectively 3611.3 and 148.4 micrograms +/- 35.9/g of creatinine. The cystathioninuric patient excreted 20 times more cystathionine mono-oxo acids in the urine than healthy subjects.

Oxidation of cystathionamine and lanthionamine by lentil seedlings amine oxidase.

Cystathionamine and lanthionamine are good substrates for lentil seedlings amine oxidase. One mole of hydrogen peroxide and one mole of ammonia per mole of substrate are produced, indicating that only one amino group is oxidized to aldehyde. The aminoaldehydes so originated undergo cyclization by intramolecular Schiff base formation. The pH optimum for the oxidation of either cystathionamine or lanthionamine is 7.0 in potassium phosphate buffer. The Km values are 0.61 and 0.84 mM respectively, similar to that for cystamine (0.8 mM).

Detection of cystathionine ketimine in bovine cerebellum.

A new sulfur-containing cyclic imino acid, cystathionine ketimine, has been detected in bovine cerebellum by gas chromatography, gas chromatography-mass spectrometry, and high pressure liquid chromatography procedures. Gas chromatography and gas-mass analyses are based on derivatization of endogenous cystathionine ketimine with diazomethane after a simple enrichment procedure. The high pressure liquid chromatography procedure takes advantage of the selective absorbance at 380 nm of the phenyl isothiocyanate-ketimine interaction product. The concentration of this new sulfur imino acid found in a pool of four bovine cerebella is approximately 0.5 nmol/g.

[35S]Lanthionine ketimine binding to bovine brain membranes.

2H-1,4-Thiazine-5,6-dihydro-3,5-dicarboxylic acid (trivial name: lanthionine ketimine) is a cyclic sulfur-containing imino acid detected in bovine brain extracts. This compound has been synthesized in a heavily labeled form starting from L-[35S]cysteine and purified by high performance liquid chromatography. We demonstrate the existence of a saturable and reversible binding of [35S]lanthionine ketimine to bovine brain membranes. A single population of binding sites with a concentration of 260 +/- 12 fmol/mg protein and a dissociation constant of 58 +/- 14 nM is present. Specific binding is competitively inhibited by other structurally similar imino acids, namely S-aminoethyl-L-cysteine ketimine and cystathionine ketimine. These results suggest a possible functional role for these ketimines in nervous system.

Influence of diet on cystathionine ketimine and lanthionine ketimine content in human urine.

A simple HPLC procedure for the routine analyses of Cystathionine ketimine (CK) and Lanthionine ketimine (LK) content in human urine has been developed. The values obtained in morning urine of fifteen healthy subjects (both sexes, 25-45 years old) on a common mixed diet are 330-2480 micrograms/g creatinine (mean 1110) of CK and 100-420 micrograms/g creatinine (mean 200) of LK. Quantitation of the two ketimines in urine of subjects on strictly vegetarian diet indicate that while the excretion of LK is independent of the diet, the excretion of CK is significantly decreased in conditions of vegetarian diet.