Simultaneous determination of methionine and total homocysteine in human plasma by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method for the simultaneous determination of methionine and total homocysteine in human plasma is described. dl-[²H₄]Methionine and dl-[²H₈]homocystine were used as internal standards. The method involved reduction of the disulfide bond with dithiothreitol, purification by cation-exchange chromatography using a BondElut SCX cartridge and derivatization with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of N(O,S)-isobutyloxycarbonyl ethyl ester (IBC-OEt) derivatives for methionine and [²H₄]methionine, respectively, and the fragment ions ([M+H–COOisoBu–COOEt]⁺) for IBC-OEt derivatives for homocysteine and [²H₄]homocysteine, respectively. The sensitivity, specificity, accuracy and precision of the method were demonstrated to be satisfactory for measuring concentrations of methionine and total homocysteine in human plasma.     

The stereochemistry of beta-lactam formation in penicillin biosynthesis.

1. (2R,3S)-[U-14C,3-3H1]- and (2R,3R)-[U-14C,2,3-3H2] Cysteine hydrochlorides have been separately synthesised. The latter compound has been shown to have uniform distributions of tritium between C-2 and C-3. 2. The abvoe cysteines and (2R)-[U-14C,3,3,3',3'-3H4]cystine have been converted to samples of penicillin G by Penicillium chrysogenum. 3. Incorporation results indicate that all but 14% of the tritium is lost from the (2R,3S)-[3-3H1]isomer; that 42% of tritium is retained by the non-stereospecifically C-3 tritiated cystine; and that 58% of tritium is retained by the (2R,3R)-[2,3-3H2]isomer on conversion to penicillin G. 4. Degradation of the penicillin G derived from (2R,3R)-[U-14C,2,3-3H2]cysteine hydrochloride has indicated that in fact about 87% of the original C-3 tritium of cysteine is retained at C-5 of penicillin G. 5. The results indicate stereospecificity in the cyclisation giving rise to the beta-lactam ring in penicillin G in nature with loss of the 3-pro-S-hydrogen and rentention of the 3-pro-R-hydrogen of cysteine. Thus there is net retention of stereochemistry in the cyclisation.     

Pharmacokinetic studies of methionine in rats using deuterium-labeled methionine quantitative assessment of remethylation

The pharmacokinetics of methionine has been studied in rats by means of stable isotope methodology. After the i.v. bolus injection of [2H7]methionine (5 mg/kg body wt.), the plasma concentrations of [2H7]methionine, demethylated [2H4]homocysteine and remethylated [2H4]methionine were determined simultaneously with endogenous methionine and homocysteine by gas chromatography-mass spectrometry. The half-life for [2H7]methionine were 35.0 +/- 6.9 min. The appearance of the metabolites, [2H4]homocysteine and [2H4]methionine, in the plasma was very rapid. The fraction of [2H7]methionine that remethylated to [2H4]methionine through [2H4]homocysteine were 0.185 +/- 0.028. The administered [2H7]methionine did not influence the plasma levels of endogenous methionine and homocysteine. The present stable isotope methodology has made it possible to evaluate the pharmacokinetics of methionine, including the estimation of remethylation.     

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%.     

Cysteine and methionine-precursors of methyl sulphone metabolites of 2,4',5-trichlorobiphenyl in mice

In order to account for the origin of the sulphur atom in methyl sulphones derived from polychlorinated biphenyls (PCB), groups of mice were treated with 2,4'-5-trichlorobiphenyl and [35S]cysteine or [35S]methionine, respectively. Control animals received the labelled amino acids only. The radioactive substances extracted from lung tissue were characterized by partition between hexane and sulphuric acid, thin-layer radiochromatography (TLRC), gas chromatography (GC) and mass fragmentography (MF). In lung extracts of both experimental groups sulphuric acid soluble metabolites were present: Their Rf-values on TLRC were identifical with that of 4-methylsulphonyl-2,4'-5-trichlorobiphenyl and their identity was confirmed by GC and GC-MF, which also indicated the presence of a minor sulphone isomer. The study shows that both cysteine and methionine could function as donors of the sulphur atom in the methyl sulphone metabolites derived from PCB.     

On the disulphide bonds of rhodopsins.

Carboxymethylation using 14C- or 3H-labelled iodoacetic acid has been used to identify the cysteine residues in bovine rhodopsin involved in the formation of the two intramolecular disulphide bridges. Iodo[2-14C]acetic acid was used to modify 5.8-5.9 residues of cysteine under non-reducing conditions. After dialysis and reduction of disulphide bridges by 2-mercaptoethanol, iodo[2-3H]acetic acid was employed to covalently modify 3.3-3.6 residues of cysteine. Peptide purification and sequencing has unambiguously shown that cysteine residues 322 and 323 are only carboxymethylated after reduction of disulphide bridges. Indirect evidence presented, now coupled with the earlier finding [Findlay & Pappin (1986) Biochem. J. 238, 625-642] suggests that the other disulphide bridge is formed between cysteine residues 110 and 187. A comparison is made of all the sequences of mammalian rhodopsins and colour pigments and attention is drawn to the fact that whereas Cys-322 and Cys-323 are conserved only in three rhodopsins (bovine, ovine and human), the residues corresponding to Cys-110 and Cys-187 are found in all the visual proteins (from rods as well as human cones).     

Biosynthesis of biopterin. Studies on the mechanism of 6-pyruvoyltetrahydropteridine synthase

[1'-3H]- and [2'-3H]dihydroneopterin triphosphate (NH2TP) were prepared enzymatically from [4-3H]- and [5-3H]glucose and converted to tetrahydrobiopterin (BH4) by an extract from bovine adrenal medulla. The formation of BH4 from both [1'-3H]- and [2'-3H]-NH2TP proceeds with virtually complete loss of the respective tritium label. The breaking of the CH-bond at C-1' is characterized by a kinetic isotope effect of 2.6 +/- 0.5. A smaller kinetic isotope effect of 1.5 +/- 0.2 was found for the breaking of the CH-bond at C-2'.     

Properties of microsomal phospholipases in rat liver and hepatoma.

Phospholipase A1, A2 and lysophospholipase activities in microsomes of Novikoff hepatoma host rat liver and regenerating rat liver were compared using 1-[9', 10'-3H2]palmitoyl-2-[1'-14C] linoleoyl-sn-glycero-3-phosphoethanolamine, 1-[1' -3H-]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and 1-[9', 10'-3H2]palmitoyl-sn-glycero-3-phosphoethanolamine as substrates. 1. Microsomes of all three tissues showed two pH dependent peaks of hydrolytic activity, one at pH 7.5 and another at pH 9.5. 2. Phospholipid hydrolytic activity in microsomes from host liver and regenerating liver require Ca2+ for hydrolysis at pH 9.5, but not at pH 7.5. Hepatoma microsomes require Ca2+ for activity at both pH values. 3. Phospholipase A1 activity, stimulated by addition of Triton X-100 to the incubation mixtures, was detected in both host liver and regenerating liver microsomes. There was no evidence of phospholipase A1 activity in hepatoma microsomes. 4. Phospholipase A2 was detected in microsomes of all three tissues using 1-[1'-3H] hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine as a substrate. The activity required calcium and was inhibited by Triton X-100. 5. Lysophospholipase activity was evident in the microsomes from all three tissues. The activity was inhibited by both Ca2+ and Triton X-100. 6. Differences were also detected between host liver and hepatoma microsomal phospholipid hydrolase activities with respect to the effect of increasing protein concentration, apparent Michaelis-Menten constants, and time course of the reaction.     

Tracer-derived total and folate-dependent homocysteine remethylation and synthesis rates in humans indicate that serine is the main one-carbon donor

Hyperhomocysteinemia in humans is associated with genetic variants of several enzymes of folate and one-carbon metabolism and deficiencies of folate and vitamins B12 and B6. In each case, hyperhomocysteinemia might be caused by diminished folate-dependent homocysteine remethylation, but this has not been confirmed in vivo. Because published stable isotopic tracer approaches cannot distinguish folate-dependent from folate-independent remethylation, we developed a dual-tracer procedure in which a [U-13C5]-methionine tracer is used in conjunction with a [3-13C]serine tracer to simultaneously measure rates of total and folate-dependent homocysteine remethylation. In young female subjects, plasma [U-13C4]homocysteine enrichment, a surrogate measure of intracellular [U-13C5]methionine enrichment, reached approximately 90% of the plasma [U-13C5]methionine enrichment. Methionine-methyl and -carboxyl group fluxes were in the range of previous reports (approximately 25 and approximately 17 micromol.kg(-1).h(-1), respectively). However, the rate of overall homocysteine remethylation (approximately 8 micromol.kg(-1).h(-1)) was twice that of previous reports, which suggests a larger role for homocysteine remethylation in methionine metabolism than previously thought. By use of estimates of intracellular [3-13C]serine enrichment based on a conservative correction of plasma [3-13C]serine enrichment, serine was calculated to contribute approximately 100% of the methyl groups used for total body homocysteine remethylation under the conditions of this protocol. This contribution represented only a small fraction (approximately 2.8%) of total serine flux. Our dual-tracer procedure is well suited to measure the effects of nutrient deficiencies, genetic polymorphisms, and other metabolic perturbations on homocysteine synthesis and total and folate-dependent homocysteine remethylation.     

The biosynthesis of ovothiol A (N-methyl-4-mercaptohistidine). Identification of S-(4'-L-histidyl)-L-cysteine sulfoxide as an intermediate and the products of the sulfoxide lyase reaction.

Crude extracts of Crithidia fasciculata catalyse the formation of 4-mercapto-L-histidine, an intermediate in the biosynthesis of ovothiol A (N1-methyl-4-mercaptohistidine), in the presence of histidine, cysteine, Fe2+ and pyridoxal phosphate. This activity was present in a 35-55% ammonium sulfate fraction that was shown to produce a transsulfuration intermediate in the absence of pyridoxal phosphate. The transsulfuration intermediate was isolated and identified as S-(4'-L-histidyl)-L-cysteine sulfoxide. The synthase activity, partially purified by anion-exchange chromatography, was shown to require oxygen and could be used to synthesize a number of isotopically labeled S-(4'-L-histidyl)-L-cysteine sulfoxides. Sulfoxide lyase activity was partially resolved from the synthase by anion-exchange chromatography. The phenylhydrazone of the product derived from the cysteine moiety of the sulfoxide coeluted with the phenylhydrazone of pyruvate on HPLC, but this assignment could not be confirmed by mass spectral analysis. S-(4'-[14C]L-histidyl)-[U-13C3,15N]L-cysteine sulfoxide was synthesized and converted to products of the lyase reaction in the presence of lactate dehydrogenase and NADH. The 13C-labeled product was identified by 13C-NMR spectroscopy as lactate and the primary product of the lyase reaction is therefore pyruvate. With S-(4'[3H]L-histidyl)-[14C]L-cysteine sulfoxide as the substrate [14C]lactate, [14C]cysteine and [3H]4-mercaptohistidine could be detected as products of the lyase reaction, but the sum of the two thiol species exceeded the amount of sulfoxide substrate used. Evidence is presented that this anomaly was due to the utilization of sulfur from dithiothreitol for the formation of cysteine.     

Identification of NAc-HCPC and NAc-beta-CEC, and qualitative analyses of sulphur amino acids in the urine of a patient with cystathioninuria using liquid chromatography/atmospheric pressure ionization mass spectrometry.

Standard sulphur amino acids and various cystathionine metabolites in the urine of a patient with cystathioninuria were analysed using liquid chromatography/mass spectrometry with an atmospheric pressure ionization interface system. Very intense quasi-molecular ions ([M + H]+) of synthetic cystathionine, N-monoacetylcystathionine, perhydro-1,4-thiazepine-3,5-dicarboxylic acid, S-(3-hydroxy-3-carboxy-n-propyl)cysteine, S-(2-carboxyethyl) cysteine, S-(2-hydroxy-2-carboxyethyl)homocysteine, S-(carboxymethyl)homocysteine, N-acetyl-S-(3-hydroxy-3-carboxy-n-propyl)cysteine and N-acetyl-S-(2-carboxyethyl)cysteine were observed by this method. Quasi-molecular ions ([M + H]+) of these sulphur amino acids were observed in the urine sample of the patient with cystathioninuria, and N-acetyl-HCPC and N-acetyl-beta-CEC as N-substituted sulphur amino acids were also identified in the urine of the same patient.     

Analysis of [2H7]methionine, [2H4]methionine,methionine, [2H4]homocysteine and homocysteine in plasma by gas chromatography–mass spectrometry to follow the fate of administered [2H7]methionine

Homocysteine plays a key role in several pathophysiological conditions. To assess the methionine–homocysteine kinetics by stable isotope methodology, we developed a simultaneous quantification method of [²H₇]methionine, [²H₄]methionine, methionine, [²H₄]homocysteine and homocysteine in rat plasma by gas chromatography–mass spectrometry (GC–MS). [¹³C]Methionine and [¹³C]homocysteine were used as analytical internal standards to account for losses associated with the extraction, derivatization and chromatography. For labeled and non-labeled homocysteine measurements, disulfide bonds between homocysteine and other thiols or proteins were reduced by dithiothreitol. The reduced homocysteine and methionine species were purified by cation-exchange chromatography and derivatized with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantification was carried out by selected ion monitoring of the molecular-related ions of N(O,S)-isobutyloxycarbonyl ethyl ester derivatives on the chemical ionization mode. The intra- and inter-day precision of the assay was less than 6% for all labeled and non-labeled methionine and homocysteine species. The method is sensitive enough to determine pharmacokinetics of labeled methionine and homocysteine.     

Folate deprivation reduces homocysteine remethylation in a human intestinal epithelial cell culture model: role of serine in one-carbon donation

Little is known about homocysteine metabolism in intestine. To address this question, we investigated homocysteine metabolism under conditions of folate adequacy and folate deprivation in the Caco-2 cell line, a model of human intestinal mucosal cells. Caco-2 cells were cultured in media enriched with [3-(13)C]serine and [U-(13)C(5)]methionine tracers, and enrichments of intracellular free amino acid pools of these amino acids as well as homocysteine, cystathionine, and cysteine were measured by using gas chromatography/mass spectrometry. Homocysteine transsulfuration plus folate-dependent and total remethylation were quantified from these amino acid enrichments. Homocysteine remethylation accounted for 19% of the intracellular free methionine pool in cells cultured with supplemental folate, and nearly all one-carbon units used for remethylation originated from the three carbon of serine via folate-dependent remethylation. Labeling of cystathionine and cysteine indicated the presence of a complete transsulfuration pathway in Caco-2 cells, and this pathway produced 13% of the intracellular free cysteine pool. Appearance of labeled homocysteine and cystathionine in culture medium suggests export of these metabolites from intestinal cells. Remethylation was reduced by one-third in folate-restricted cell cultures (P < 0.001), and only approximately 50% of the one-carbon units used for remethylation originated from the three carbon of serine under these conditions. In conclusion, the three carbon of serine is the primary source of one-carbon units used for homocysteine remethylation in folate-supplemented Caco-2 cell cultures. Remethylation is reduced as a result of folate restriction in this mucosal cell model, and one-carbon sources other than the three carbon of serine contribute to remethylation under this condition.     

高蛋氨酸饲料喂养的大鼠高同型半胱氨酸血症模型的实验研究

探讨高蛋氨酸饲料诱导高同型半胱氨酸血症的适应剂量及其对相关代谢的影响.选择质量分数1%、2%和3%蛋氨酸饲料喂饲大鼠,高压液相色谱法测定血清同型半胱氨酸、半胱氨酸和还原形谷胱甘肽含量.结果表明饲料中1%蛋氨酸可以使血清同型半胱氨酸水平升高且无生长抑制等毒副作用,2%和3%蛋氨酸饲料喂养大鼠后出现摄食量减少和生长抑制等毒...     

Agarose-selected variants of two human tumor cell lines exhibit altered methionine auxotrophy

Our aim was to determine if the selection of human tumor cells with enhanced anchorage-independent growth capacity was associated with alterations in methionine auxotrophy. Cells with an increased ability to form colonies on soft agarose were selected from human melanoma (MeWo) and neuroepithelioma (SK-N-MC) cell lines. In contrast to their respective parental lines, a high proportion of the agarose-selected variants were completely unable to proliferate in methionine-free medium containing its immediate precursor homocysteine. The variants exhibited no significant change in their total DNA 5-methylcytosine content and showed no stimulation of either RNA or DNA synthesis upon the addition of homocysteine when the cells were cultured in methionine-free medium. These variants were unable to synthesize [3H]S-adenosylmethionine from [3H]adenine and homocysteine. The failure to detect the accumulation of [3H]S-adenosylmethionine in these variant lines was not likely due to the enhanced turnover of S-adenosylmethionine but rather to a reduced ability to synthesize methionine from homocysteine and 5-methyltetrahydrofolic acid. These results support our hypothesis that alterations in the metabolism of methionine and/or intracellular transmethylating activities may contribute to, or be associated with, the autonomous growth of malignant human tumor cells.     

Stereospecificity of sodium borohydride reduction of tyrosine decarboxylase from Streptococcus faecalis.

Sodium boro[3H]hydride reduction of tyrosine decarboxylase from Streptococcus faecalis followed by complete hydrolysis of the enzyme produces epsilon-[3H]pyridoxyllysine. Degradation of this material to [4'-3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase shows that the re side at C-4' of the cofactor is exposed to solvent at pH 5.5 and 7.0. After binding of L-tyrosine at pH 5.5 or tyramine at pH 7.0 to the holoenzyme, sodium boro[3H]hydride reduction proceeds from the si face at C-4' of the substrate . cofactor complex. This indicates one of two conformational changes occurs upon binding of substrate; either rotation about the C-4 to C-4' bond in the cofactor or rotation about the axis through the C-5 and C-5' bond.     

Synthesis of some newer derivatives of substituted quinazolinonyl-2-oxo/thiobarbituric acid as potent anticonvulsant agents

5-[1'-[3"-Aminoacetyl-2"-methyl-6",8"-dihalosubstitutedquinazolin-4"(3"H)-onyl]-thiosemicarbazido]-2-oxo/thiobarbituric acids 3a-3h and 5-[2'-amino-5'-[3"-aminomethylene-2"-methyl-6",8"-dihalosubstitutedquinazolin-4"(3"H)-onyl]-1',3',4'-thiadiazol-2'-yl]-2-oxo/thiobarbituric acid 5a-5h were prepared by incorporating 1-[3'-aminoacetyl-2'-methyl-6",8"-dihalosubstituted-quinazolin-4'(3'H)-onyl]-thiosemicarbazides 2a-2d and 2-amino-5-[3'-aminomethylene-2'-methyl-6',8'-dihalosubstituted-quinazolin-4'(3'H)-onyl]-1,3,4-thiadiazoles 4a-4 h respectively at 5(th) position of 2-oxo/thiobarbituric acids (via Mannich reaction). All the newly synthesized compounds were screened for their anti-convulsant activity in MES and PTZ models and were compared with standard drugs phenytoin sodium and sodium valproate. Interestingly, these compounds were found to be devoid of sedative and hypnotic activities when tested. Out of the compounds studied, the most active compound 5h, that is 5-[2'-amino-5'-[3"-aminomethylene-2"-methyl-6",8"-dibromoquinazolin-4"(3"H)-onyl]-1',3',4'-thiadiazol-2'-yl]-2-thiobarbituric acid showed activity (90%) more potent than the standard drug.     

Determination of D- and L-enantiomers of methionine and [2H3]methionine in plasma by gas chromatography-mass spectrometry

A method for the stereoselective determination of D- and L-enantiomers of both methionine and [(2)H3]methionine in rat plasma was developed using gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS-SIM). DL-[(2)H7]Methionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The amino acids were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. Endogenous L-methionine concentrations in 50 microl of rat plasma were measured with relative intra- and inter-day precision of 4.0 and 6.3%, respectively. The intra- and inter-day reproducibility in the amounts of D- and L-[(2)H3]methionine determined were in good agreement with actual amount added.     

Deoxyribose ring conformation of [d(GGTATACC)]2: an analysis of vicinal proton-proton coupling constants from two-dimensional proton nuclear magnetic resonance

Exchangeable and nonexchangeable protons of [d(GGTATACC)]2 in aqueous cacodylate solution were assigned from two-dimensional nuclear Overhausser effect (2D NOE) spectra. With phase-sensitive COSY and double quantum filtered COSY (DQF-COSY) experiments, the cross-peaks resulting from deoxyribose ring conformation sensitive proton-proton vicinal couplings, i.e., all 1'-2', 1'-2", 2'-3', and 3'-4' couplings and six from 2"-3' couplings, were observed. From the cross-peak fine structure, the 2',2" proton assignments can be confirmed; coupling constants J1'2' and J1'2" and sums of coupling constants involving H2' and H2" for all residues and H3' for C8 were obtained. The DISCO procedure [Kessler, H., Muller, A., & Oschkinat, H. (1985) Magn. Reson. Chem. 23, 844-852] was used to extract individual 1'-2' and 1'-2" coupling constants. The sum of coupling constants involving H1' or H3' was measured from the one-dimensional spectrum where signal overlap is not a problem. Analysis of the resulting coupling constants and sums of coupling constants, in the manner of Rinkel and Altona [Rinkel, L. J., & Altona, C. (1987) J. Biomol. Struct. Dyn. 4, 621-649], led to the following conclusion: C2'-endo deoxyribose ring conformation is predominant for every residue, but a significant amount of C3'-endo conformation may exist, ranging from 14% to 30%.