Unusual metabolism of sulfur-containing amino acids in rats treated with DL-propargylglycine

S-(2-Hydroxy-2-carboxyethyl)homocysteine, S-(3-hydroxy-3-carboxy-n-propyl)-cysteine, N-acylated S-(beta-carboxyethyl)cysteine, and N-acylated S-(3-hydroxy-3-carboxy-n-propyl) cysteine were excreted in the urine after DL-propargylglycine treatment. Cystathionine was also accumulated in several tissues of DL-propargylglycine-treated rats. N-Monoacetylcystathione was found in the liver of rats and was also detected in the kidney and serum. Cystathionine gamma-lyase activity in liver decreased to about 4% of that of control rats 24 h after the DL-propargylglycine injection, and alanine aminotransferase activity decreased to about 35% of that of control rats. On the other hand, aspartate aminotransferase and cystathionine beta-synthese activity did not show significant changes from those of control rats. The ability of normal tissues to synthesize cystathionine utilizing cystathionine beta-synthase was 1.98 +/- 0.40 mumol/min/g in liver, 0.61 +/- 0.13 in kidney, and 0.18 +/- 0.015 in brain. The maximal contents of cystathionine in rat tissues and the administered amounts of DL-propargylglycine agreed well with the ability to synthesize cystathionine in each tissue.     

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.     

Monitoring exposure to acrylamide by the determination of S-(2-carboxyethyl)cysteine in hydrolyzed hemoglobin by gas chromatography-mass spectrometry

Acrylamide is a potent cumulative neurotoxin in animals and man. In vivo exposure to this electrophile results in the formation of a covalently bound reaction product with cysteine residues in hemoglobin. This adduct yields on acid hydrolysis S-(2-carboxyethyl)cysteine which has been analyzed by capillary gas chromatography with mass spectrometry. Globin isolated from the blood of rats exposed to acrylamide was spiked with an internal standard (globin treated in vitro with d3-acrylamide) and was then hydrolyzed with 6 N HCl. The protein hydrolysate was fractionated on a Dowex 50W H+ ion exchange column and the amino acids in the partially purified extract were determined as N-heptafluorobutyryl methyl esters using an OV-1701 fused silica capillary column. Quantitation was made by chemical ionization (isobutane) selective ion monitoring in which the ions m/z 386 (M-OCH3)+ derived from derivatized S-(2-carboxyethyl)cysteine in the sample and the corresponding ion m/z 389 from the added deuterium-labeled internal standard were monitored. The dose-response relationship between production of hemoglobin adduct and dose of acrylamide (0.1 mg/kg-5 mg/kg) is curved, showing an increasing slope with increasing doses of acrylamide.     

Simultaneous determination of urinary cystathionine, lanthionine, and their cyclic compounds using liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization

A measurement system for cystathionine (Cysta) lanthionine (LT), and (AEC), and reduced products of their ketimines, perhydro-1,4-thiazapine-3,5-dicarboxylic acid (PHTZDC), 1,4-thiomorpholine-3,5-dicarboxylic acid (TMDA) and 1,4-thiomorpholine-3-carboxylic acid (TMA) in the urine samples of a patient with cystathioninuria and normal human subjects has been developed, using column liquid chromatography-mass spectrometry. The recoveries were about 90–105% for Cysta, LT and AEC, and about 77–87% for PHTZDC, TMDA and TMA after ion-exchange treatment. The concentrations of Cysta and PHTZDC in the urine of a patient with cystathioninuria were much higher compared with those in the urine of normal human subjects. The concentrations of AEC and TMDA were almost the same. LT and TMA could not be detected in the urine samples by this method. This method proved useful for the determination of sulfur-containing amino acids and their cyclic compounds in biological samples.     

Effect of regulatory mutations of sulphur metabolism on the levels of cysteine- and homocysteine-synthesizing enzymes in Neurospora crassa

1. Regulation of four enzymes involved in cysteine and homocysteine synthesis, i.e. cysteine synthase (EC 4.2.99.8), homocysteine synthase (EC 4.1.99.10), cystathionine beta-synthase (EC 2.1.22) and gamma-cystathionase (EC 4.4.1.1) was studied in the wild type and sulphur regulatory mutants of Neurospora crassa. 2. Homocysteine synthase and cystathionine beta-synthase were found to be regulatory enzymes but only the former is under control of the cys-3 - scon system regulating several enzymes of sulphur metabolism, including gamma-cystathionase. 3. The results obtained with the mutants strongly suggest that homocysteine synthase plays a physiological role as an enzyme of the alternative pathway of methionine synthesis. Cysteine synthase activity was similar in all strains examined irrespective of growth conditions. 4. The sconc strain with derepressed enzymes of sulphur metabolism showed an increased pool of sulphur amino acids, except for methionine. Particularly characteristic for this pool is a high content of hypotaurine, a product of cysteine catabolism.     

Identification of a novel selenium metabolite, Se-methyl-N-acetylselenohexosamine, in rat urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry and--electrospray ionization tandem mass spectrometry

The major urinary metabolite of selenium (Se) in rats was identified by HPLC-inductively coupled argon plasma mass spectrometry (ICP-MS) and--electrospray tandem mass spectrometry (ESI-MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI-MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]-) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-beta-D-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine.     

Mass spectrometric quantification of endogenous beta-endorphin

Fast atom bombardment (FAB) mass spectrometry and multiple reaction monitoring (MRM) in the B/E linked-field scan mode were used to quantify endogenous beta-endorphin (BE) in individual human pituitary extracts. The experimental protocol includes the addition of a stable isotope-labeled internal standard ((2H4-Ile22)BE1-31, human) to the tissue homogenate before extraction, purification of the native BE by a combination of Sep-Pak chromatography and gradient high-performance liquid chromatography (HPLC), trypsin digestion to cleave BE into smaller peptides, and separation of the tryptic fragment BE20-24 (NAIIK) by isocratic reversed-phase HPLC. Mass spectrometric quantification is based upon recording either (a) the [M + H]+ ions of NAIIK and its deuterated analog ((2H4)NAIIK), or (b) the transitions ([NAIIK + H](+)----[NAI]+) and [((2H4)NAIIK + H](+)----[(2H4)NAI]+) using the B/E linked-field scan. Linear calibration curves were obtained using these two mass spectrometric techniques from standard solutions containing 1.25-20 micrograms of BE; each standard solution also contained 10 micrograms of (2H4)BE. The amounts (means +/- s.d.) of endogenous BE in five separate human pituitaries were found to be 156 +/- 84 [( M + H]+ method) and 169 +/- 99 pmol mg-1 protein (MRM method).     

Simultaneous determination of fatty, dicarboxylic and amino acids based on derivatization with isobutyl chloroformate followed by gas chromatography-positive ion chemical ionization mass spectrometry

Gas chromatography-mass spectrometry (GC-MS) with positive ion chemical ionization (PICI) using isobutane as reagent gas was applied for analysis of isobutoxycarbonyl/isobutyl derivatives of 13 fatty, 6 dicarboxylic and 13 amino acids in a single run. For all investigated compounds (except several amino acids) the quasimolecular ions [MH](+) were registered. Asparagine underwent fragmentation via decarboxylation followed by elimination of OC(4)H(9) ([M-117](+)), whereas serine and tyrosine produced the cluster ions [M+C(4)H(9)OCO](+). Estimated detection limits were 6-250 pg in the total ion current (TIC) mode and 3-10 times lower using the selected-ion monitoring (SIM) mode.     

Liquid chromatographic—mass spectrometric analysis for screening of patients with cystinuria, and identification of cystine stone

Analyses of amino acids in the urine of a normal human and of patients with heterozygous and homozygous cystinuria have been carried out, using liquid chromatography—mass spectrometry with an atmospheric pressure ionization interface system. A kidney cystine stone was also analysed by this system. Very intense quasi-molecular ions ([M + H]⁺) of standard cystine, arginine, lysine and ornithine were observed on mass chromatograms as base peaks. Mass chromatograms of the urine samples from a normal human and from patients with heterozygous and homozygous cystinuria were easily distinguishable. The retention times in the mass chromatogram and mass spectrum of kidney stone cystine was almost the same as that of authentic cystine.     

The role of amino acids in the regulation of hydrogen sulfide production during ultradian respiratory oscillation of Saccharomyces cerevisiae

We previously demonstrated that periodic H2S production during aerobic continuous culture of Saccharomyces cerevisiae resulted in ultradian respiratory oscillation, and that H2S production was dependent on the activity of sulfate uptake and the level of sulfite. To investigate the mechanism of regulation of the sulfate assimilation pathway and of respiratory oscillation, several amino acids were pulse-injected into cultures during respiratory oscillation. Injection of sulfur amino acids or their derivatives perturbed respiratory oscillation, with changes in the H2S production profile. Four major regulators of H2S production in the sulfate assimilation pathway and respiratory oscillation were identified: (1) O-acetylhomoserine, not O-acetylserine, as a sulfide acceptor, (2) homoserine/threonine as a regulator of O-acetylhomoserine supply, (3) methionine/S-adenosyl methionine as a negative regulator of sulfate assimilation, and (4) cysteine (or its derivatives) as an essential regulator. The results obtained after the addition of DL-propargylglycine (5 microM and 100 microM) and cystathionine (50 microM) suggested that the intracellular cysteine level and cystathionine gamma-lyase, rather than methionine/S-adenosylmethionine, play an essential role in the regulation of sulfate assimilation and respiratory oscillation. Based on these results and those of our previous reports, we propose that periodic depletion of cysteine (or its derivatives), which is involved in the detoxification of toxic materials originating from respiration, causes periodic H2S production.     

Genomic basis of cystathioninuria (MIM 219500) revealed by multiple mutations in cystathionine gamma-lyase (<Emphasis Type="Italic">CTH</Emphasis>)

Hereditary cystathioninuria (MIM 219500) is presumed to be caused by deficiency of the activity of cystathionine gamma-lyase (cystathionase; CTH EC 4.4.1.1), which is normally required for the conversion of methionine into cysteine. To date, no mutations have been described among patients with cystathioninuria. From genomic DNA, we sequenced CTH in four unrelated probands with cystathioninuria. We found two nonsense mutations, namely exon 8 c.940-941delCT and exon 11 c.1220delC, and two missense mutations, namely exon 2 c.356C>T (T67I) and exon 7 c.874C>G (Q240E). All affected subjects were either simple homozygotes or compound heterozygotes. A common non-synonymous single nucleotide polymorphism in exon 12, namely c.1364G>T (S403I), was also identified and characterized in four ethnic groups. The reagents described in this report make the molecular diagnosis of cystathioninuria possible, allowing for studies of phenotype-genotype correlation. Also, the availability of a common non-synonymous SNP can allow for testing of association of the CTH gene with biochemical traits affected by trans-sulfuration, such as plasma concentrations of homocysteine or even cystathionine itself, in addition to more downstream clinical phenotypes, such as vascular disease.     

Analysis of cyclic and acyclic analogs of retinol, retinoic acid, and retinal by laser desorption ionization-, matrix-assisted laser desorption ionization-mass spectrometry, and UV/Vis spectroscopy.

Laser desorption ionization (LDI)- and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry (LDI-MS, MALDI-MS) at 337-nm laser wavelength were used to analyze retinol (vitamin A), retinoic acid, and retinal and their analogs 3-hydroxyretinol, 3-hydroxyretinoic acid, 3-hydroxyretinal, 4-oxoretinol, 4-oxoretinoic acid, 4-oxoretinal, 3,4-didehydroretinol (vitamin A(2)), 3,4-didehydroretinoic acid, 3,4-didehydroretinal, acycloretinol, acycloretinoic acid, and acycloretinal. The compounds exhibit sufficient ionizability which allows to obtain mass spectra by LDI which are similar in quality to those obtained by MALDI. Mass spectra were recorded with a linear time-of-flight (TOF) instrument or a reflectron-type (RETOF) instrument in positive-ion mode. Under the conditions of LDI-MS the compounds form abundant radical molecular ions (M+*), whereas in the MALDI mass spectra abundant protonated molecular ions ([M + H]+) are observed. Characteristic fragment ions provide additional structural information. High-performance liquid chromatography (HPLC) coupled with UV/Vis photodiode detection was used to assist in retinoid characterization. Synthesis of 3-hydroxyretinal, 4-oxoretinal, and acycloretinal was performed by oxidative cleavage of the all-trans-carotenoids of zeaxanthin, canthaxanthin, and lycopene.     

Mass spectrometry in the study of bioclasters of amino acids and peptides]

The study of homo- and heterocluster quasimolecular ions of 20 L-amino acids (A) and five dipeptides by the TOF-PDMS method indicated that the intensity of quasimolecular ions of the corresponding homo-([An + H]+ and [Bm + H]+, where A and B are biomolecules (A, dipeptides), n and m = 1 .... 5) and heteroclusters ([An.Bm + H]+, n and m = 1 .... 5) depends mainly on the hydrophobicity of the constituents of the A cluster. The most intensive peaks of homo- and heterocluster ions were obtained for hydrophobic amino acids: L-Ile, L-Leu, L-Val, and L-Phe, and for dipeptides containing these amino acids. The assumption is made that the stereochemical parameters of heterocluster quasimolecular ions in the TOF-PDMS method are determined by the physicochemical mechanisms involved in the processes of ionization/desorption of biomolecules and do not reflect directly biologically significant interactions of biomolecules in vivo.     

CAD MIKES: a new method for a rapid and unequivocal structural identification of organic acids in biological fluids. A first application to a case of methylmalonic aciduria

A novel application of Collisionally Activated Decomposition Mass analysed Ion Kinetic Energy (CAD MIKE) spectrometry to separation and positive structural identification of urinary methylmalonic acid (MMA) (the pathognomonic compound for the diagnosis of methylmalonic acidurias) is presented. CAD MIKES scans of EI ionic species at m/z 119 ([M + H]+) and m/z 101 ([M-OH]+) have been obtained from a pure standard of MMA and from crude urinary acid fractions. With reference to the procedures employed so far, the advantages of the proposed method lie in fast and simplified sample pretreatment and in a quick non-controversial response to a clinical suspicion of serious, life-threatening inherited metabolic diseases.     

Nutritional factors affecting nisin production by Lactococcus lactis subsp. actis NIZO 22186 in a synthetic medium

L. DE VUYST. 1995 A minimal synthetic medium (SM8) for nisin-producing Lactococcus lactis subsp. lactis strains has been designed; it consists of eight growth-stimulating amino acids (glutamic acid, methionine, valine, leucine, threonine, arginine, isoleucine and histidine), five vitamins (biotin, calcium pantothenate, nicotinic acid, pyridoxine and riboflavin) and the mineral salts dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, magnesium sulphate and trisodium citrate. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, cysteine or cystathionine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.     

Reversible cyclization ofS-(2-oxo-2-carboxyethyl)-L-homocysteine to cystathionine ketimine

Summary S-(2-oxo-2-carboxyethyl)homocysteine (OCEHC), produced by the enzymatic monodeamination of cystathionine, is known to cyclize producing the seven membered ring of cystathionine ketimine (CK) which has been recognized as a cystathionine metabolite in mammals. Studies have been undertaken in order to find the best conditions of cyclization of synthetic OCEHC to CK and for the preparation of solid CK salt product. It has been found that ring closure takes place at alkaline pH and is highly accelerated in 0.5 M phosphate buffer. The sodium salt of CK has been prepared by controlled additions of NaOH to water-ethanol solution of OCEHC under N₂ atmosphere. A solid product is obtained which, dissolved in water, shows the spectral features of CK. Solutions of the sodium salt of CK show the presence of a pH depending reversible equilibrium with the open OCEHC form. Plot of the absorbance at 296 nm in function of pH indicates that at pH 9 the compound is completely cyclized while at pH 6 is totally in the open OCEHC form. At intermediate pHs variable ratios between the two forms occur. According to the results obtained by the spectral analysis, HPLC assays of the sodium salt of CK show different patterns depending on the pH of the elution buffer.Abbreviations CK cystathionine ketimine - OCEHC S-(2-oxo-2-carboxyethyl) homocysteine - HPLC high performance liquid chromatography     

Covalent binding of 4-nitrobenzyl mercaptan S-sulfate to the sulfhydryl groups of hepatic cytosolic proteins and bovine serum albumin with mixed disulfide bond formation

4-Nitrobenzyl [35S]mercaptan S-sulfonic acid ([35S]NBM S-sulfate), a new type of reactive metabolite of the thiol [35S]NBM in rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate, bound rapidly and covalently at pH 7.4 and 37 degrees C to the sulfhydryl groups of rat liver cytosolic proteins with formation of disulfide bonds. From the radioactive proteins was isolated and identified the sole amino acid adduct, S-([35S]NBM)cysteine, after their acid hydrolysis under the anaerobic conditions. Bovine serum albumin (BSA), a model protein with a single SH group, also reacted readily with radioactive NBM S-sulfate to form a disulfide bond in stoichiometric manner. S-([35S]NBM)-cysteine was also isolated and identified as the sole amino acid adduct from the well-washed, radioactive BSA after the same anaerobic acid hydrolysis. A normal hepatic level of GSH not only retarded the BSA-NBM adduct formation completely, but also detached the radioactivity from BSA by the reduction of the disulfide bond with formation of [35S]NBM and its disulfide. Of twenty-one amino acids examined at pH 7.4 and 37 degrees C, only cysteine reacted with NBM S-sulfate and afforded S-(NBM)cysteine with concomitant formations of S-sulfocysteine, cystine, NBM, and its disulfide.     

Mutations affecting the sulphur assimilation pathway in Aspergillus nidulans: their effect on sulphur amino acid metabolism

Several sul-reg mutants of Aspergillus nidulans isolated as constitutive for arylsulphatase were studied with respect to the regulation of enzymes involved in cysteine and homocysteine synthesis and to the pool of sulphur amino acids. All mutants examined showed a decreased concentration of glutathione as compared with the wild type, and all mutants, with one exception, had a decreased total pool of sulphur amino acids. The results suggest that the mutants are leaky in the sulphate assimilation pathway. They show derepression of cysteine synthase, homocysteine synthase, cystathionine beta-synthase and gamma-cystathionase. In spite of having derepressed homocysteine synthase, the enzyme which constitutes an alternative pathway for homocysteine synthesis, the sul-reg mutations do not suppress lesions in genes required for the main homocysteine-synthesizing pathway. This indicates that the derepression of homocysteine synthase is not in itself sufficient for physiological functioning of this enzyme, but seems to depend also on the effectiveness of cysteine synthesis and sulphide formation.     

Determination of L-cysteine in amino acid mixture and human urine by flow-injection analysis with a biamperometric detector.

Based on the electrocatalytic oxidation of cysteine at pretreated platinum electrode and the flow-injection biamperometry for irreversible couple, a novel electrochemical detector is proposed for the selective determination of cysteine in amino acid mixtures and human urine samples. A thin-layer flow through cell was used to achieve large electrode surface area to volume ratio. Two identical pretreated platinum electrodes were mounted in the cell with an applied potential difference of 10 mV. By coupling two independent and irreversible electrode processes, namely, the oxidation of cysteine and the reduction of platinum oxide, the biamperometric detection scheme has been established. The resulting current is linear to cysteine over the range 4 x 10(-7)-4 x 10(-5) M with the detection limit 1 x 10(-7) M (15 pmol). The selectivity of the detector is tested by 55 foreign species including 26 ions, 11 amino acids, 6 vitamins, and 12 other compounds possibly found in urine. The detector performs well as a routine assay, showing high efficiency (180 samples/h) and good reproductivity shown by a RSD of 0.6% for eight repeated determinations of 2 x 10(-6) M cysteine. The urine samples are detected directly without the need of pretreatment or adding other reagents.     

PLP-dependent H(2)S biogenesis

The role of endogenously produced H(2)S in mediating varied physiological effects in mammals has spurred enormous recent interest in understanding its biology and in exploiting its pharmacological potential. In these early days in the field of H(2)S signaling, large gaps exist in our understanding of its biological targets, its mechanisms of action and the regulation of its biogenesis and its clearance. Two branches within the sulfur metabolic pathway contribute to H(2)S production: (i) the reverse transsulfuration pathway in which two pyridoxal 5'-phosphate-dependent (PLP) enzymes, cystathionine β-synthase and cystathionine γ-lyase convert homocysteine successively to cystathionine and cysteine and (ii) a branch of the cysteine catabolic pathway which converts cysteine to mercaptopyruvate via a PLP-dependent cysteine aminotransferase and subsequently, to mercaptopyruvate sulfur transferase-bound persulfide from which H(2)S can be liberated. In this review, we present an overview of the kinetics of the H(2)S-generating reactions, compare the structures of the PLP-enzymes involved in its biogenesis and discuss strategies for their regulation. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.