Presence Of Semicarbazide Research Articles

The presence of semicarbazide in crustaceans collected from natural habitats in Thailand

Semicarbazide (SEM) has been used as a marker residue of the banned veterinary drug nitrofurazone (NFZ). Although evidence indicates that SEM can be found in some natural crustaceans that have never been exposed to NFZ, such information is limited to a few species. The present study aimed to investigate the natural occurrence of SEM in wild crustaceans in Thailand. A total of 14 species, all economically important food animals, were captured from different regions of Thailand. Tissue-bound SEM and its parent drug NFZ were determined by the UPLC-MS/MS and LC-MS methods, respectively. The results showed that while NFZ was not detected in any samples, the tissue-bound SEM could be found in every natural crustacean species investigated. However, the prevalence and concentration varied greatly. The occurrence of SEM in the freshwater palaemonid Macrobrachium prawns is generally much higher than in the marine penaeid shrimps/prawns. SEM was found in 33% and 80% of the giant river prawn (Macrobrachium rosenbergii) muscles (<0.10–0.46 ng/g) and shells (3.68–13.22 ng/g), respectively. In contrast, SEM was not detected in the muscles of penaeid shrimps/prawns (with few exceptions), but it was occasionally found in the shells at low levels (usually <1 ng/g). The shells of saltwater crabs also contained higher levels of SEM than the muscles. For instance, the highest SEM levels detected in the mud crab (Scylla sp.) muscles and shells were 0.40 and 22.75 ng/g, respectively. However, the situation was reversed for the rice-field crab (Sayamia sp. and Esanthelphusa sp.), in which SEM was not detected in all shells but detected in the muscles (up to 1.46 ng/g). The fact that SEM is often found in wild crustaceans implies a natural origin of this substance. Consequently, using SEM as a marker residue of NFZ is controversial and should be reconsidered.

Green-Electrochemical Synthesis of Novel Catechol Derivative Based on O-Quinone Nucleophilic Addition

In this work, a green-electrochemical synthesis was applied on oxidation of catechol (1) to o -benzoquinone (2) using cyclic voltammetry and controlled potential coulometry. Encouraging results were obtained and has been investigated in the presence of semicarbazide ( 3 ) as a nucleophile in an ethanol/phosphate buffer mixture (pH 7.0). The results showed that novel heterocyclic compound, triazinone, functionalized with hydroxyl and amide groups have been synthesised. The effect of pH, number of cycles, and scan rate were studied to attain a good yield and a high purity of products. The results indicate that the generated o -quinone ( 2 ) participates in a Michael addition reaction with nucleophilic hydrazide via EC mechanism to form the new triazinone adduct. After purification, the products have been characterized by IR, UV-Visible, 1 H and 13 C NMR methods. The green-electrochemical synthesis approach produced novel organics with excellent purities and yields while being environmentally friendly and free of hazardous reagents.

Why semicarbazide (SEM) is not an appropriate marker for the usage of nitrofurazone on agricultural animals

A comprehensive global database on semicarbazide (SEM) in foodstuffs and food ingredients is presented, with over 4000 data collected in foods such as seafood (crustaceans, fish powders), meat (beef, chicken powders), dairy products (e.g. raw milk, milk powders, whey, sweet buttermilk powder, caseinate, yoghurt, cheese), honey and other ingredients. The results provide evidence that the presence of SEM in certain dairy ingredients (whey, milk protein concentrates) is a by-product of chemical reactions taking place during the manufacturing process. Of the dairy ingredients tested (c. 2000 samples), 5.3% showed traces of SEM > 0.5 µg/kg. The highest incidence of SEM-positive samples in the dairy category were whey (powders, liquid) and milk protein concentrates (35% positive), with up to 13 µg/kg measured in a whey powder. Sweet buttermilk powder and caseinate followed, with 27% and 9.3% positives, respectively. SEM was not detected in raw milk, or in yoghurt or cheese. Of the crustacean products (shrimp and prawn powders) tested, 44% were positive for SEM, the highest value measured at 284 µg/kg. Fish powders revealed an unexpectedly high incidence of positive samples (25%); in this case, fraudulent addition of shellfish shells or carry-over during processing cannot be excluded. Overall, the data provide new insights into the occurrence of SEM (for dairy products and fish powders), substantially strengthening the arguments that SEM in certain food categories is not a conclusive marker of the use of the illegal antibiotic nitrofurazone.

Properties of a semicarbazide-sensitive amine oxidase in human umbilical artery.

The metabolism of some aromatic amines by amine oxidase activities in human umbilical artery homogenates has been studied. The inhibitory effects of clorgyline showed that 5-hydroxytryptamine (5-HT) and tryptamine, 1 mM, were predominantly substrates for monoamine oxidase (MAO) type A, whereas MAO-A and B were both involved in the metabolism of beta-phenylethylamine (PEA), 100 microM, and tyramine, 1 mM. About 20-30% of tyramine and PEA metabolism was resistant to 1 mM clorgyline, but sensitive to inhibition by semicarbazide, 1 mM, indicating the presence of a semicarbazide-sensitive amine oxidase (SSAO). Benzylamine, 1 mM, appeared to be metabolized exclusively by SSAO with a Km (161 microM) at pH 7.8 similar to that found for SSAO in other human tissues. Tyramine and PEA were relatively poor substrates for SSAO, with very high apparent Km values of 17.6 and 13.3 mM, respectively, when determined in the presence of clorgyline, 10(-3) M, added to inhibit any metabolism of those amines by MAO activities. However, kinetic studies with benzylamine indicated that clorgyline, 10(-3) M, also appears to inhibit SSAO competitively such that the true Km values for tyramine and PEA may be about 60% of those apparent values given above. No evidence for the metabolism of 5-HT or tryptamine by SSAO was obtained. The aliphatic amine methylamine was recently shown to be a specific substrate for SSAO in umbilical artery homogenates. We have used benzylamine and methylamine as SSAO substrates in histochemical studies to localize SSAO in tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Modelling the roles of MAO and SSAO in glucose transport

Amine oxidase substrates such as benzylamine and methylamine have been shown to stimulate glucose uptake by increasing the recruitment of the glucose transporter GLUT4 from vesicles within the cell to the cell surface. Inhibition of this effect by the presence of semicarbazide and catalase led to the suggestion that the process is mediated by the H(2)O(2) produced in the oxidation of these amines. Tyramine, which is a substrate for both MAO and SSAO, can also stimulate this process and in that case both MAO and SSAO inhibitors attenuate the effect. Benzylamine does not occur physiologically and tyramine is normally present in only very low amounts. We have suggested that adrenaline, which also stimulates glucose metabolism through adrenoceptors, may act as the physiological substrate for GLUT4 recruitment. It is a substrate for MAO but not SSAO. However, oxidation of adrenaline by MAO releases both H(2)O(2) and methylamine for further oxidation by SSAO. In order to gain a fuller understanding of this process we have performed simulation studies that may be used to assess the contributions of the amine oxidases to the process under a variety of conditions. The results are consistent with the experimentally observed behaviour. This approach not only helps to establish the feasibility of this process but also allows behaviour prediction and the identification of further experimental approaches.

Integrated Rate Equation Considering Product Inhibition and Its Application to Kinetic Assay of Serum Ethanol

Kinetic assay of serum ethanol was investigated by predicting maximal product absorbance at 340 nm (A(mk)) through fitting to the yeast alcohol dehydrogenase reaction curve with the integrated rate equation, taking into account product inhibition in the presence of semicarbazide. Predicted A(mk) linearly responded to the preset constant of steady-state concentration of acetaldehyde (C(ald)). An exponential correlation function was established between desired C(ald) and putative A(mk) for authentic ethanol. For unknown samples, iterative fitting to reaction curve till preset constant C(ald) and resultant A(mk) satisfied this exponential correlation function yielded A(mk) with variation coefficient <4.3%. Variations in enzyme activity, data range and kinetic parameters showed negligible effects. The recovery was consistent to 100% with resistance to methanol and isopropanol. The upper limit of linear response for A(mk) was about 40 times of the lower limit. These results indicated that this kinetic method was reliable for serum ethanol assays with obvious advantages.

Semicarbazide: occurrence in food products and state-of-the-art in analytical methods used for its determination

This review provides an overview of the information currently available about the presence of semicarbazide (SEM) in food. Likely sources of SEM in food matrices are summarised and discussed. Detailed information is given about the analytical methods used to determine SEM; features and drawbacks associated with them are carefully evaluated. Performance criteria characterising the analytical methods discussed are also given.

Spectrophotometric Assays for l-Lysine α-Oxidase and γ-Glutamylamine Cyclotransferase

A new assay for l-lysine α-oxidase is described. In this assay, the oxidized product generated from l-lysine is reacted with semicarbazide to form α-keto-ϵ-aminocaproate semicarbazone. Formation of the α-keto acid semicarbazone is continuously monitored spectrophotometrically at 248 nm (ϵ 10,160 ± 240 M−1 cm−1). The method was adapted to provide a new assay for γ-glutamylamine cyclotransferase. This enzyme catalyzes the conversion of many l-γ-glutamylamines to 5-oxo-l-proline and free amine. A biologically important substrate is Nϵ-(γ-l-glutamyl)-l-lysine, which is converted to 5-oxo-l-proline and l-lysine by the action of γ-glutamylamine cyclotransferase. The l-lysine generated from Nϵ-(γ-l-glutamyl)-l-lysine in an endpoint assay is converted to α-keto ϵ-aminocaproate semicarbazone in the presence of semicarbazide, excess l-lysine α-oxidase, and catalase. The methods were applied to the determination of γ-glutamylamine cyclotransferase activity of partially purified preparations of the bovine kidney enzyme and to detect γ-glutamylamine cyclotransferase activity in rat kidney and liver homogenates.

Role of Semicarbazide-sensitive Amine Oxidase on Glucose Transport and GLUT4 Recruitment to the Cell Surface in Adipose Cells

The previous characterization of an abundant population of non-adrenergic imidazoline-I2 binding sites in adipocytes and the recent demonstration of the interplay between these binding sites and amine oxidases led us to analyze the amine oxidase activity in membranes from isolated rat adipocytes. Adipocyte membranes had substantial levels of semicarbazide-sensitive amine oxidase (SSAO). SSAO activity and immunoreactive SSAO protein were maximal in plasma membranes, and they were also detectable in intracellular membranes. Vesicle immunoisolation analysis indicated that GLUT4-containing vesicles from rat adipocytes contain substantial levels of SSAO activity and immunoreactive SSAO protein. Immunotitration of intracellular GLUT4 vesicles indicated that GLUT4 and SSAO colocalize in an endosomal compartment in rat adipocytes. SSAO activity was also found in GLUT4 vesicles from 3T3-L1 adipocytes and rat skeletal muscle. Benzylamine, a substrate of SSAO activity, caused a marked stimulation of glucose transport in isolated rat adipocytes in the presence of very low vanadate concentrations that by themselves were ineffective in exerting insulin-like effects. This synergistic effect of benzylamine and vanadate on glucose transport was totally abolished in the presence of semicarbazide, a specific inhibitor of SSAO. Subcellular membrane fractionation revealed that the combination of benzylamine and vanadate caused a recruitment of GLUT4 to the plasma membrane of adipose cells. The stimulatory effects of benzylamine and vanadate on glucose transport were blocked by catalase, suggesting that hydrogen peroxide production coupled to SSAO activity plays a crucial regulatory role. Based on these results we propose that SSAO activity might contribute through hydrogen peroxide production to the in vivo regulation of GLUT4 trafficking in adipose cells.

Inhibitory Effect of some carbazides on corrosion of aluminium in hydrochloric acid and sodium hydroxide solutions

AbstractThe dissolution of aluminium in hydrochloric acid and sodium hydroxide solutions in the presence of semicarbazide, thiosemicarbazide and sym.diphenylcarbazide as corrosion inhibitors has been studied using thermometric, weight‐loss and polarization methods. The three methods gave consistent results. The higher inhibition efficiency of these compounds in acidic than in alkaline madia may be due to the less negative potential of aluminium in hydrochloric acid solution, favouring adsorption of the additive. The adsorption of these compounds were found to obey Frumkin adsorption isotherm.Cathodic polarization measurements showed that these compounds are cathodic inhibitors and their adsorption in the double layer does not change the mechanism of the hydrogen evolution reaction. The results are analysed in terms of both molecular and cationic adsorption.

Deuterium D(V/K) Isotope Effects on Ethanol Oxidation in Hepatocytes: Importance of the Reverse ADH‐Reaction

The kinetic deuterium isotope effect, D(V/K), on ethanol oxidation was measured on hepatocytes from rat and pig by the radiometric competitive method using 14C-labelled ethanol containing deuterium in the (1-R)-position. The corrected D(V/K) values of 2.68 and 2.80 for rat and pig hepatocytes respectively were significantly different, suggesting differences in the amount of non-ADH ethanol oxidizing activity. The apparent isotope effects declined rapidly with time when acetaldehyde was present in the medium as a result of the reduction to ethanol of the [14C]-acetaldehyde formed from the double labelled ethanol by alcohol dehydrogenase (ADH). Fructose and cyanamide caused the acetaldehyde concentration during ethanol oxidation to increase by entirely different mechanisms, and the isotope effect to decrease with time, as did also the addition of acetaldehyde. The apparent first order rate constant for the reverse ADH reaction, assuming the reactants to be acetaldehyde and the ADH-NADH complex, was determined by two methods giving comparable results. In the presence of semicarbazide, which removes acetaldehyde, the isotope effect was nearly constant. This was the case also when the acetaldehyde concentration was very low (less than 1 microM) for other reasons, as in hepatocytes from starved animals. A mathematical formula describing the expected decrease of the apparent isotope effect with time was derived. The different response of pig and rat hepatocytes to addition of fructose (the 'fructose effect') is suggested to be caused by differences in activity of aldehyde dehydrogenases in the two species.

Conversion of canavanine to α-keto-γ-guanidinooxybutyrate and to vinylglyoxylate and 2-hydroxyguanidine

It was observed previously that hydroxyguanidine is formed in the reaction of canavanine(2-amino-4-guanidinooxybutanoate) with amino acid oxidases. The present work shows that hydroxyguanidine is formed by a nonenzymatic β,-γ-elimination reaction following enzymatic oxidation at the α-C and that the abstraction of the β-H is general-base catalyzed. The elimination reaction requires the presence in the α-position of an anion-stabilizing group—the protonated imino group (iminium ion group) or the carbonyl group. The iminium ion group is more activating than the carbonyl group. Elimination is further facilitated by protonation of the guanidinooxy group. The other product formed in the elimination reaction was identified as vinylglyoxylate (2-oxo-3-butenoate), a very highly electrophilic substance. The product resulting from hydrolysis following oxidation was identified as α-keto-γ-guanidinooxybutyrate (ketocanavanine). The ratio of hydroxyguanidine to ketocanavanine depended upon the concentration and degree of basicity of the basic catalyst and on pH. In the presence of semicarbazide, the elimination reaction was prevented because the imino group in the semicarbazone derivative of ketocanavanine is not significantly protonated. Incubation of canavanine with 5′-deoxypyridoxal also yielded hydroxyguanidine. Since the elimination reactions take place under mild conditions, they may occur in vivo following oxidation at the α-C of l-canavanine (ingested or formed endogenously) or of other amino acids with a good leaving group in the γ-position (e.g., S-adenosylmethionine, methionine sulfoximine, homocyst(e)ine, or cysteine-homocysteine mixed disulfide) by an l-amino acid oxidase, a transaminase, or a dehydrogenase. Therefore, vinylglyoxylate may be a normal metabolite in mammals which at elevated concentrations may contribute to the in vivo toxicity of canavanine and of some of the other above-mentioned amino acids.

Evidence for an aldehyde intermediate in the catalytic mechanism of thiamine oxidase

Thiamine oxidase catalyzes the four-electron oxidation of the 5-hydroxyethyl group of thiamine to form thiamine acetic acid via an aldehyde intermediate. Evidence for the formation of this intermediate is derived from a number of kinetic approaches. The rate of thiamine acetic acid formation, as monitored by the rate of proton release, is subject to substrate inhibition and to inhibition by the presence of semicarbazide while the rate of O 2 consumption (due to thiamine oxidation to the aldehyde and subsequently to the carboxylic acid) is unaffected. The transient formation of an intermediate with a maximal absorption at 370 nm in stopped-flow turnover experiments is dependent on the pH and the substrate concentration, and is prevented by the presence of semicarbazide, thus suggesting this transient absorption intermediate to be a result of formation of the aldehyde intermediate. A similar spectral intermediate is observed when hydroxythiamine is the substrate but is not observed with pyrithiamine. In the presence of large concentrations of pyrithiamine, the enzyme undergoes an irreversible inactivation which is not reversed on removal of pyrithiamine or its oxidation products by gel filtration or dialysis. This inhibition is prevented by the presence of thiols or of semicarbazide and is suggested to be due to the release of the aldehyde form of pyrithiamine from the catalytic site, which then reacts with the enzyme in a nonspecific manner. The structure of the 370-nm-absorbing intermediate is currently unknown but is suggested not to be the “yellow form” of thiamine. This suggestion is due to observed differences in absorption spectral properties and to the fact that it can also be formed from hydroxythiamine, which does not form the “yellow form” of thiamine on alkaline treatment. Taken together, these data suggest that, at or below saturating concentrations, thiamine remains bound to the catalytic site during the two sequential two-electron transfer steps, with 2 mol O 2 being reduced to 2 mol H 2O 2. At high concentrations (>10 K m ), the intermediate thiamine aldehyde can be displaced from the catalytic site by thiamine simply by a mass-action effect.

Comparison of three methods for the elimination of the labile fraction of HbA 1

Three different procedures were used to remove the "labile" fraction in the chromatographic quantitation of hemoglobin A1 (HbA1) by a minicolumn assay: (a) preincubation of the erythrocytes at 37 degrees C in isotonic saline for 4 h, (b) preincubation in the presence of semicarbazide-aniline at pH 5.0 for 30 min, and (c) preincubation in acetate buffer at pH 5.5 for 30 min. The results show that the two latter methods are not only more rapid but are also slightly more effective. The use of the acetate buffer is preferred because this reagent is more easily prepared and also because the presence of semicarbazide and aniline did not markedly accelerate the dissociation of Hb pre-A1c at pH 5.5. The procedure relies simply on the greater instability of Schiff base in acidic solution. There is a significant correlation between the "labile" fraction and the plasma glucose concentration at sampling time. The results support the view that the elimination of the "labile" precursor is essential to preserve the utility of the assay.

Enzyme-coupled measurement of ethanol in whole blood and plasma with a centrifugal analyzer.

We describe an automated enzymic method for ethanol determination with a centrifugal analyzer (the GEMSAEC) by measuring the rate of the reaction catalyzed by alcohol dehydrogenase and coupled to aldehyde dehydrogenase. The detection limits, reproducibility, and accuracy of the method have been evaluated. It can be applied to whole blood or plasma, with or without previous deproteinization. Our results, compared with those by an automated alcohol dehydrogenase method in the presence of semicarbazide, show an improved linearity, sensitivity, and rapidity of determination.

Co-operative mutagenic actions of bisulfite and nitrogen nucleophiles

Abstract The combined effect of bisulfite and a nitrogen nucleophile, i.e. semicarbazide, methoxyamine or hydroxylamine, to chemically modify cytosine and to cause mutation and inactivation of bacteriophage lambda was investigated. A rapid transamination of cytidine with each of the amines took place in the presence of bisulfite, and the reaction product was solely the N (4)-transaminated 5,6-dihydrocytidine-6-sulfonate. Modifications of cytidine with bisulfite alone and with the nitrogen nucleophile alone were much slower reactions than those using a combination of bisulfite and the nucleophile. Whereas the product of the modification with the bisulfite/semicarbazide, 5,6-dihydro-4-semicarbazido-2-ketopyrimidine ribofuranoside-6-sulfonate, is convertible to 4-semicarbazido-2-ketopyrimidine ribofuranoside by treatment with a phosphate buffer, the products of the modification with the bisulfite/methoxyamine and with the bisulfite/hydroxylamine, i.e. 4-methoxy-5,6-dihydrocytidine-6-sulfonate and 4-hydroxy-5,6-dihydrocytidine-6-sulfonate, were stable in phosphate buffer. Inactivation and the “clear” mutation of bacteriophage lambda were observed when the phage was treated with sodium bisulfite in the presence of semicarbazide, methoxyamine or hydroxylamine. Under the conditions used, only very small increases in the mutation frequency were obtained by treatment of the phage with bisulfite alone or with the base alone. It was concluded that the residues, 5,6-dihydro-4-semicarbazido-2-ketopyrimidine-6-sulfonate, 4-methoxy-5, 6-dihydrocytosine-6-sulfonate and 4-hydroxy-5,6-dihydrocytosine-6-sulfonate in DNA are the causes of the mutation. When phage that had been inactivated by the semicarbazide/bisulfite reagent was subsequently treated with a phosphate buffer, a reactivation took place. The rate of the reactivation increased as the concentration of phosphate in the buffer increased. This reactivation was not accompanied by change in the mutation frequency. No reactivation was observed after a similar incubation when the prior inactivation had been induced by either methoxyamine/bisulfite or hydroxylamine/bisulfite. These results indicate that the 4-semicarbazido-2-ketopyrimidine residue is also mutagenic but is less lethal than the corresponding 5,6-dihydro-6-sulfonate structure. These results offer the first clear example of the co-operative mutagenic action of two different reagents.

Role of formaldehyde in the utilization of C1 compounds via the ribulose monophosphate cycle

AbstractFormaldehyde, an intermediate in methanol oxidation, plays an important role in controlling methanol utilization by microorganisms which fix carbon via the ribulose monophosphate (RMP) cycle. The extracellular formaldehyde concentration profiles in batch cultures at low and high starting methanol concentrations with and without the presence of semicarbazide in the media, suggest the role played by formaldehyde. The impact of formaldehyde on growth is demonstrated by the time dependent and the initial methanol concentration‐dependent cell‐mass‐yield coefficient. Kinetic studies of the enzymes involved in the oxidation and incorporation of C1 units suggest that enzyme inhibition cannot account for the observed growth inhibition. A mechanism is proposed to explain methanol and formaldehyde utilization that stresses the transcriptional regulation of mass flow around formaldehyde in assimilation and oxidation pathways through repression and induction.

Co-operative mutagenic actions of bisulfite and nitrogen nucleophiles.

The combined effect of bisulfite and a nitrogen nucleophile, i.e. semicarbazide, methoxyamine or hydroxylamine, to chemically modify cytosine and to cause mutation and inactivation of bacteriophage lambda was investigated. A rapid transamination of cytidine with each of the amines took place in the presence of bisulfite, and the reaction product was solely the N(4)-transaminated 5,6-dihydrocytidine-6-sulfonate. Modifications of cytidine with bisulfite alone and with the nitrogen nucleophile alone were much slower reactions than those using a combination of bisulfite and the nucleophile. Whereas the product of the modification with the bisulfite/semicarbazide, 5,6-dihydro-4-semicarbazido-2-ketopyrimidine ribofuranoside-6-sulfonate, is convertible to 4-semicarbazido-2-ketopyrimidine ribofuranoside by treatment with a phosphate buffer, the products of the modification with the bisulfite/methoxyamine and with the bisulfite/hydroxylamine, i.e. 4-methoxy-5,6-dihydrocytidine-6-sulfonate and 4-hydroxy-5,6-dihydrocytidine-6-sulfonate, were stable in phosphate buffer. Inactivation and the “clear” mutation of bacteriophage lambda were observed when the phage was treated with sodium bisulfite in the presence of semicarbazide, methoxyamine or hydroxylamine. Under the conditions used, only very small increases in the mutation frequency were obtained by treatment of the phage with bisulfite alone or with the base alone. It was concluded that the residues, 5,6-dihydro-4-semicarbazido-2-ketopyrimidine-6-sulfonate, 4-methoxy-5, 6-dihydrocytosine-6-sulfonate and 4-hydroxy-5,6-dihydrocytosine-6-sulfonate in DNA are the causes of the mutation. When phage that had been inactivated by the semicarbazide/bisulfite reagent was subsequently treated with a phosphate buffer, a reactivation took place. The rate of the reactivation increased as the concentration of phosphate in the buffer increased. This reactivation was not accompanied by change in the mutation frequency. No reactivation was observed after a similar incubation when the prior inactivation had been induced by either methoxyamine/bisulfite or hydroxylamine/bisulfite. These results indicate that the 4-semicarbazido-2-ketopyrimidine residue is also mutagenic but is less lethal than the corresponding 5,6-dihydro-6-sulfonate structure. These results offer the first clear example of the co-operative mutagenic action of two different reagents.

Enzymatic reactions of methionine sulfoximine. Conversion to the corresponding alpha-imino and alpha-keto acids and to alpha-ketobutyrate and methane sulfinimide.

L-Methionine sulfoximine is a substrate of L-amino acid oxidase (Crotalus adamanteus), glutamine transaminase, and gamma-cystathionase. In the reaction catalyzed by L-amino acid oxidase, methionine sulfoximine is converted to aplph-imino-gamma-methylsulfoximinylbutyrate, which undergoes rapid gamma elimination yielding methane sulfinimide and 2-imino-3-butenoic acid. Methane sulfinimide is converted to methane sulfonamide, methane sulfinic acid, and methane sulfonic acid; 2-imino-3-butenoic acid is hydrolyzed to vinylglyoxylate, which polymerizes to an insoluble product. When the reaction is carried out in the presence of semicarbazide, the imine formed initially is quantitatively trapped as alpha-keto-gamma-methylsulfoximinylbutyrate semicarbazone, from which the free alpha-keto acid may be obtained. When the reaction is carried out in the presence of a mercaptan (RSH), a gamma exchange reaction occurs leading to formation of a new alpha-keto acid substituted in the gamma position by an SR-group; thus, alpha-keto-gamma-(beta-hydroxyethiol)butyric acid (S-(hydroxyethyl)-2-keto-4-mercaptobutyric acid) was obtained when L-methionine sulfoximine was oxidized in the presence of 2-mercaptoethanol, and enzymatic transamination of this alpha-keto acid with L-glutamine gave the new amino acid, L-omega-hydroxyethionine. The reaction of L-methionine sulfoximine with gamma-cystathionase yields 1 mol each of alpha-ketobutyrate and methane sulfinimide; the latter is hydrolyzed almost exclusively to methane sulfinic acid. Transamination of L-methionine sulfoximine yields the corresponding alpha-keto acid (alpha-keto-gamma-methylsulfoximinylbutyrate), which is stable. Some of these reactions may occur in vivo, and thus contribute to the toxicity of L-methionine sulfoximine.

Comparative studies on human plasma monoamine oxidase in normal subjects and in fibrotic liver disease

Plasma monoamine oxidase from normal human subjects and from a patient with hemosiderosis and hepatic fibrosis were purified to 200-fold. Kinetic properties were studied at various pH's and in the presence of different inhibitors. pH studies showed that the two enzyme preparations have different pH optimum ranges, although the maximum activity occurs at the same pH (7.2). At both acidic and basic pH's, abnormal monoamine oxidase showed lower percent activity. Inhibition of the enzyme by semicarbazide revealed that abnormal monoamine oxidase lost more activity than normal monoamine oxidase at all inhibitor concentrations. The apparent K m 's of the two were significantly different in the presence of semicarbazide. Kynuramine was found to act as an inhibitor to benzylamine oxidation at the enzyme concentrations used in this study. At optimum substrate concentration, it inhibits both preparations similarly, but at low substrate concentrations the inhibition on abnormal monoamine oxidase is more dependent on substrate concentration. The inhibition by semicarbazide and kynuramine are distinctly different. This suggests that two distinct inhibitory sites may be involved in this enzyme. These observations indicate that plasma monoamine oxidase from a patient with hepatic fibrosis has different intrinsic molecular properties than normal human plasma monoamine oxidase. Thus, elevated plasma monamine oxidase activity in liver disease may not be simply due to an increase of the enzyme normally found, but rather represents the presence of isoenzymes of monoamine oxidase.