Body condition prepartum and its association with term placentome nutrient transporters, one­carbon metabolism pathway activity, and intermediate metabolites in Holstein cows.

We investigated linkages among BCS prior to calving and placentome concentrations of metabolites, proteins in one­carbon metabolism (OCM) and protein synthesis, and nutrient transport. Multiparous Holstein cows retrospectively divided by prepartal BCS at -4 weeks relative to parturition into high BCS (HBCS = 3.58 ± 0.23; n = 9) or normal BCS (NBCS = 3.02 ± 0.17; n = 13) were used. BCS was assessed using a 5-point scale (1 = thin, 5 = fat). Four placentomes per cow were collected at delivery and frozen in liquid N. Western blotting was used for protein abundance. Cystathionine-ß-synthase (CBS) and betaine-homocysteine-S-methyltransferase (BHMT) activity were measured via 14C assays. Amino acids (AA) and metabolites in OCM were measured by liquid chromatography mass spectrometry (LC-MS). Compared with NBCS cows, the cellular stress sensor p-eIF2α was more than 2-fold greater (P = 0.04) in HBCS. Abundance of the AA-catabolism enzyme branched-chain α-ketoacid dehydrogenase complex was lower (P = 0.05) in HBCS cows. Although BHMT activity did not differ, greater concentration of betaine (P = 0.01) and lower (P = 0.05) concentration of dimethylglycine in HBCS cows suggested reduced flux through the methionine cycle. Despite a lack of difference in CBS activity, lower concentrations of cystathionine (P = 0.03) and hypotaurine (P = 0.04) along with lower cysteine and the tendency for lower total GSH (P = 0.10) in HBCS cows suggested a decrease in transsulfuration. Overall, associations between OCM in placentomes and BCS at calving exist. Identifying mechanisms responsible for these effects merits further research.

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.).

In vivo 1 H MRS detection of cystathionine in human brain tumors.

PURPOSE: To report the technical aspects of noninvasive detection of cystathionine in human brain glioma with edited MRS, and to investigate possible further acquisition improvements for robust quantification of this metabolite. METHODS: In vivo 1 H MR spectra were acquired at 3 T in 15 participants with an isocitrate dehydrogenase-mutated glioma using a MEGA-PRESS (MEscher GArwood point resolved spectroscopy) sequence previously employed for 2-hydroxyglutarate detection (TR = 2 s, TE = 68 ms). The editing pulse was applied at 1.9 ppm for the edit-on condition and at 7.5 ppm for the edit-off condition. To evaluate the editing efficiency, spectra were acquired in 1 participant by placing the editing pulse for the edit-on condition at 1.9, 2.03, and 2.16 ppm. Cystathionine concentration was quantified using LCModel and a simulated basis set. To confirm chemical shifts and J-coupling values of cystathionine, the 1 H NMR cystathionine spectrum was measured using a high-resolution 500 MHz spectrometer. RESULTS: In 12 gliomas, cystathionine was observed in the in vivo edited MR spectra at 2.72 and 3.85 ppm and quantified. The signal intensity of the cystathionine resonance at 2.72 ppm increased 1.7 and 2.13 times when the editing pulse was moved to 2.03 and 2.16 ppm, respectively. Cystathionine was not detectable in normal brain tissue. CONCLUSION: Cystathionine can be detected in vivo by edited MRS using the same protocol as for 2-hydroxyglutarate detection. This finding may enable a more accurate, noninvasive investigation of cellular metabolism in glioma.

A disrupted transsulphuration pathway results in accumulation of redox metabolites and induction of gametocytogenesis in malaria.

Intra-erythrocytic growth of malaria parasite is known to induce redox stress. In addition to haem degradation which generates reactive oxygen species (ROS), the parasite is also thought to efflux redox active homocysteine. To understand the basis underlying accumulation of homocysteine, we have examined the transsulphuration (TS) pathway in the parasite, which is known to convert homocysteine to cysteine in higher eukaryotes. Our bioinformatic analysis revealed absence of key enzymes in the biosynthesis of cysteine namely cystathionine-ß-synthase and cystathionine-γ-lyase in the parasite. Using mass spectrometry, we confirmed the absence of cystathionine, which is formed by enzymatic conversion of homocysteine thereby confirming truncation of TS pathway. We also quantitated levels of glutathione and homocysteine in infected erythrocytes and its spent medium. Our results showed increase in levels of these metabolites intracellularly and in culture supernatants. Our results provide a mechanistic basis for the long-known occurrence of hyperhomocysteinemia in malaria. Most importantly we find that homocysteine induces the transcription factor implicated in gametocytogenesis namely AP2-G and consequently triggers sexual stage conversion. We confirmed this observation both in vitro using Plasmodium falciparum cultures, and in vivo in the mouse model of malaria. Our study implicates homocysteine as a potential physiological trigger of gametocytogenesis.

Synthesis and evaluation of L-cystathionine as a standard for amino acid analysis.

L-Cystathionine is a key nonprotein amino acid related to metabolic conditions. The quantitative determination of L-cystathionine in physiological fluids by amino acid analysis is important for clinical diagnosis; however, certified reference material for L-cystathionine with satisfactory purity, content, and quantity has been unavailable until recently. Consequently, a practical and simple method for the preparation of L-cystathionine was examined, which involves thioalkylation of N-tert-butoxycarbonyl-L-cysteine tert-butyl ester, derived from L-cystine, with (2S)-2-(tert-butoxycarbonyl)amino-4-iodobutanoic acid tert-butyl ester, derived from L-aspartic acid, to obtain L-cystathionine with protecting groups, followed by single-step deprotection under mild conditions. This method produces L-cystathionine in high purity (99.4%) and having sufficient percentage content according to amino acid analysis, which could be used as a standard for the amino acid analysis of physiological fluids.

Differential response of methionine metabolism in two grain legumes, soybean and azuki bean, expressing a mutated form of Arabidopsis cystathionine γ-synthase.

Methionine (Met) is a sulfur-containing amino acid that is essential in mammals and whose low abundance limits the nutritional value of grain legumes. Cystathionine γ-synthase (CGS) catalyzes the first committed step of Met biosynthesis, and the stability of its mRNA is autoregulated by the cytosolic concentration of S-adenosyl-l-methionine (SAM), a direct metabolite of Met. The mto1-1 mutant of Arabidopsis thaliana harbors a mutation in the AtCGS1 gene that renders the mRNA resistant to SAM-dependent degradation and therefore results in the accumulation of free Met to high levels in young leaves. To manipulate Met biosynthesis in soybean and azuki bean, we introduced the AtCGS1 mto1-1 gene into the two grain legumes under the control of a seed-specific glycinin gene promoter. Transgenic seeds of both species accumulated soluble Met to levels at least twice those apparent in control seeds. However, the increase in free Met did not result in an increase in total Met content of the transgenic seeds. In transgenic azuki bean seeds, the amount of cystathionine, the direct product of CGS, was markedly increased whereas the total content of Met was significantly decreased compared with control seeds. Similar changes were not detected in soybean. Our data suggest that the regulation of Met biosynthesis differs between soybean and azuki bean, and that the expression of AtCGS1 mto1-1 differentially affects the metabolic stability of sulfur amino acids and their metabolites in the two grain legumes.

The role of amino acids in spina bifida.

Our objective was to measure amniotic fluid amino acid concentrations in pregnant women diagnosed as having fetuses with spina bifida in the second trimester of pregnancy. Fifteen pregnant women who had fetuses with spina bifida detected by ultrasonography (spina bifida group) in the second trimester and 19 women who had abnormal triple screenings indicating an increased risk for Down's syndrome but had healthy fetuses (control group) were enrolled in the study. Amniotic fluid was obtained by amniocentesis. The chromosomal analysis of the study and control groups was normal. Levels of free amino acids were measured in amniotic fluid samples using EZ: fast kits (EZ: fast GC/FID free (physiological) amino acid kit) by gas chromatography (Focus GC AI 3000 Thermo Finnigan analyzer). The mean levels of alanine, cystathionine, cysteine, phenylalanine, tryptophane, and tyrosine amino acids were found to be significantly higher in fetuses of the control group than in the spina bifida group (p<0.05). The detection of significantly higher amino acid concentrations in the amniotic fluid of healthy fetuses suggests loss of amino acids from the fetus through the spinal cord may contribute to the etiology of spina bifida.

RP-HPLC method for quantitative determination of cystathionine, cysteine and glutathione: An application for the study of the metabolism of cysteine in human brain.

The RP-HPLC method for a simultaneous separation and quantitation of the dinitrophenyl derivative of cystathionine (N,N'-di-DNP) in biological samples together with GSH, GSSG, cysteine and cystine, provides a very useful tool for investigation of the transsulfuration pathway in biological samples, at the same providing results which reflect the redox status (GSH/GSSG ratio) and the potential of the generation of H2S. An application of the method for the study of the process of transsulfuration in various human brain regions shows the presence of cystathionine in all the investigated regions; it also demonstrates that cystathionine levels vary greatly between particular regions. The highest level in the thalamus and the lowest in the cerebellum were associated with respectively a low or high γ-cystathionase activity, and at the same time, a high cysteine and GSH level in the thalamus and a low value in the cerebellum. Based on the above results, one may suggest a regulatory mechanism responsible for inhibition of the CGL activity at high concentration values of cysteine and/or GSH. Simultaneous determinations of GSH and GSSG levels allow for determining the GSH/GSSG ratio, which reflects tissue redox status. The method may be also employed in determining the activity of γ-cystathionase and cystathionine-ß synthase.

Label-free high-throughput screening via mass spectrometry: a single cystathionine quantitative method for multiple applications.

Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. MS permits the selective detection of enzyme substrates or products in a wide range of biological matrices without need for derivatization, labeling, or capture technologies. As part of a cardiovascular drug discovery effort aimed at finding modulators of cystathionine beta-synthase (CBS), we used the RapidFire((R)) label-free high-throughput MS (HTMS) technology to develop a high-throughput screening (HTS) assay for CBS activity. The in vitro assay used HTMS to quantify the unlabeled product of the CBS reaction, cystathionine. Cystathionine HTMS analyses were carried out with a throughput of 7 s per sample and quantitation over a linear range of 80-10,000 nM. A compound library of 25,559 samples (or 80 384-well plates) was screened as singlets using the HTMS assay in a period of 8 days. With a hit rate of 0.32%, the actives showed a 90% confirmation rate. The in vitro assay was applied to secondary screens in more complex matrices with no additional analytical development. Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project.

Molecular and enzymatic characterization of betaC-S lyase in Streptococcus constellatus.

BACKGROUND/AIMS: Streptococcus anginosus and Streptococcus constellatus are frequently isolated from dental abscesses and other suppurative lesions. We previously reported that betaC-S lyase from a strain of S. anginosus produced significantly more hydrogen sulfide than betaC-S lyases from other streptococci. The purpose of this study was to establish the molecular and enzymatic features of the betaC-S lyase in S. constellatus and to elucidate whether this unique capacity is common to many strains of S. constellatus and S. anginosus. METHODS: The capacity of crude extract to produce hydrogen sulfide was evaluated among 16 strains of S. constellatus, S. anginosus, and Streptococcus gordonii. The lcd gene encoding betaC-S lyase was cloned from the genomic DNA of each strain to compare the deduced amino acid sequences. The recombinant betaC-S lyases of three representative strains were purified and characterized. RESULTS: Incubation of crude extracts from all strains of S. constellatus and S. anginosus with l-cysteine resulted in the production of a large amount of hydrogen sulfide. The primary sequence of betaC-S lyase was very similar among strains of S. constellatus and S. anginosus. The kinetic properties of the betaC-S lyases purified from S. constellatus resembled those for betaC-S lyases purified from S. anginosus. In contrast, the betaC-S lyases of S. constellatus and S. gordonii differed in terms of their hydrogen sulfide production, with the former producing much more. CONCLUSION: A high level of hydrogen sulfide production, which appears to be a common feature in both S. constellatus and S. anginosus, may be associated with their abscess formation.

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.

Measurement of intracellular sulfur amino acid metabolism in humans.

Methionine metabolism forms homocysteine via transmethylation. Homocysteine is either 1) condensed to form cystathionine, which is cleaved to form cysteine, or 2) remethylated back to methionine. Measuring this cycle with the use of isotopically labeled methionine tracers is problematic, because the tracer is infused into and measured from blood, whereas methionine metabolism occurs inside cells. Because plasma homocysteine and cystathionine arise from intracellular metabolism of methionine, plasma homocysteine and cystathionine enrichments can be used to define intracellular methionine enrichment during an infusion of labeled methionine. Eight healthy, postabsorptive volunteers were given a primed continuous infusion of [1-13C]methionine and [methyl-2H(3)]methionine for 8 h. Enrichments in plasma methionine, [13C]homocysteine and [13C]cystathionine were measured. In contrast to plasma methionine enrichments, the plasma [13C]homocysteine and [13C]cystathionine enrichments rose to plateau slowly (rate constant: 0.40 +/- 0.03 and 0.49 +/- 0.09 h(-1), respectively). The enrichment ratios of plasma [13C]homocysteine to [13C]methionine and [13C]cystathionine to [13C]methionine were 58 +/- 3 and 54 +/- 3%, respectively, demonstrating a large intracellular/extracellular partitioning of methionine. These values were used to correct methionine kinetics. The corrections increase previously reported rates of methionine kinetics by approximately 40%.

Is the increase in serum cystathionine levels in patients with liver cirrhosis a consequence of impaired homocysteine transsulfuration at the level of gamma-cystathionase?

BACKGROUND: It has been suggested that the major metabolic block in the methionine catabolic pathway in cirrhotics exists at the level of the enzyme S-adenosylmethionine synthetase because in previous studies using conventional amino-acid analyzers, no intermediates of transmethylation/transsulfuration were found to accumulate in plasma downstream of S-adenosylmethionine synthesis. We therefore measured serum concentration intermediates of methionine transmethylation/transsulfuration using an improved gas chromatography/mass spectrometry technique. METHODS: Serum concentrations of methionine, homocysteine, cystathionine, N,N-dimethylglycine, N-methylglycine, methylmalonic acid, 2-methylcitric acid and alpha-aminobutyric acid were determined by gas chromatography/mass spectrometry in 108 consecutive patients with liver cirrhosis at Child stages A (mild cirrhosis, n = 27) and B/C (severe cirrhosis, n = 81), 18 outpatients with non-cirrhotic liver disease, and 55 healthy individuals. RESULTS: Serum levels of methionine, N,N-dimethylglycine, N-methylglycine, cystathionine, and homocysteine were significantly higher in patients at Child stages B/C compared with those of healthy controls (P < 0.01), and they were also significantly higher than in patients with non-cirrhotic liver disease (P < 0.01 and P < 0.05 for homocysteine, respectively). They also correlated with the Child-Pugh score (P < 0.01). Homocysteine, cystathionine, N,N-dimethylglycine, N-methylglycine, methylmalonic acid, and 2-methylcitric acid correlated with serum creatinine. The mean cystathionine concentration was significantly higher in patients with creatinine > or = 1.4 mg/dl than in patients with normal creatinine values (P < 0.01). However, the differences between cirrhotics and healthy controls were still significant after correcting for creatinine. CONCLUSIONS: Our data provides indirect evidence for two hitherto unrecognized alterations of methionine metabolism in cirrhotics, i.e. impairment of the transsulfuration of homocysteine at the level of cystathionine degradation and a shift in remethylation of homocysteine towards the betaine-homocysteine-methyltransferase reaction.

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.

Simultaneous determination of cystathionine and NAc-cystathionine using liquid chromatography atmospheric pressure chemical ionization mass spectrometry.

A method for simultaneous determination of cystathionine and NAc-cystathionine in biological samples has been developed by using column liquid chromatography-mass spectrometry. The concentrations of cystathionine and NAc-cystathionine in urine, several tissues, and serum of propargylglycine-treated rats with experimental cystathioninuria were determined by scanning the [M + H]+ ions of each compound. The recoveries of authentic compounds were 95.69 +/- 4.44% (n = 10) for cystathionine and 91.59 +/- 5.26% (n = 10) for NAc-cystathionine after ion-exchange resin treatment. The contents of cystathionine and NAc-cystathionine in the urine of a patient with cystathioninuria were also measured. The results coincided well with those obtained using an amino acid analyzer.

Determination of sulphur amino acids by gas chromatography with flame photometric detection.

A selective and sensitive method has been developed for the determination of sulphur amino acids by gas chromatography (GC). Sulphur amino acids were converted into their N(S)-isopropoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection using a DB-17 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of sulphur amino acids were ca. 0.5-1 pmol per injection, and the calibration curves were linear in the range 0.5-10 nmol for each sulphur amino acid. This method was successfully applied to small urine samples without prior clean-up, and sulphur amino acids in these samples could be analysed without any influence from coexisting substances. Overall recoveries of sulphur amino acids added to urine samples were 85-113%. The analytical results of free sulphur amino acid contents in urine samples of normal subjects are presented.