S-carboxymethyl-L-cysteine Research Articles

Anti-Inflammatory Actions of Expectorants in a Rat Carrageenan-Induced Footpad Edema Model.

S-Carboxymethyl-L-cysteine (SCMS) exhibits sputum-regulating and anti-inflammatory actions. Previous studies reported the anti-inflammatory effects of SCMS on chronic inflammatory diseases, but no study has examined these effects on acute inflammatory diseases. In this study, we investigated the anti-inflammatory effects of SCMS in a rat carrageenan-induced footpad edema model, which is routinely used as an acute inflammation model. Expectorants were administered to rats with footpad edema induced by subcutaneously administering 1%λ-carrageenan to the footpad of the left posterior limb, and the dose dependency of the anti-inflammatory effects was evaluated. As a result, even when the dose of SCMS was increased to 400 mg/kg, there were no inhibitory effects on edema. Furthermore, we examined the inhibitory effects of other expectorants (ambroxol hydrochloride, N-acetyl-L-cysteine, L-cysteine ethylester hydrochloride, and L-cysteine methylester hydrochloride), which were reported to exhibit anti-inflammatory effects on chronic inflammation, on edema. However, none of these expectorants inhibited edema.

The S-oxidation of S-carboxymethyl-L-cysteine in hepatic cytosolic fractions from BTBR and phenylketonuria enu1 and enu2 mice

Mice that were heterozygous dominant for the enu1 and enu2 mutation in phenylalanine monooxygenase/phenylalanine hydroxylase (PAH) resulted in hepatic PAH assays for S-carboxymethyl-L-cysteine (SCMC) that had significantly increased calculated Km (wild type (wt)/enu1, 1.84–2.12 fold increase and wt/enu2 a 2.75 fold increase in PAH assays). The heterozygous dominant phenotypes showed a significantly reduced catalytic turnover of SCMC (wt/enu1, 6.11 fold decrease and wt/enu2 an 11.25 fold decrease in calculated Vmax). Finally, these phenotypes also had a significantly reduced clearance, CLE (wt/enu1, 13.02 fold and wt/enu2, a 30.80–30.94 fold decrease) The homozygous recessive phenotype (enu1/enu1) was also found to have significantly increased calculated Km (2.16 fold increase), a significantly reduced calculated Vmax (11.35–12.33 fold decrease) and CLE (24.75–25.00 fold decrease). The enu2/enu2, homozygous recessive phenotype had no detectable PAH activity using SCMC as substrate.The identity of the enzyme responsible for the C-oxidation of L-phenylalanine (L-Phe) and the S-oxidation of SCMC in wt/wt (BTBR) mice was identified using monoclonal antibody and selective chemical inhibitors and was found to be PAH.This in vitro mouse hepatic cytosolic fraction metabolism investigation provides further evidence to support the hypothesis that an individual possessing one variant allele for PAH will result in a poor metaboliser phenotype that is unable to produce significant amounts of S-oxide metabolites of SCMC.

Mouse recombinant phenylalanine monooxygenase and the S‐oxygenation of thioether substrates

The substrate specificity of mouse recombinant phenylalanine monooxygenase (mPAH) has been investigated with respect to the mucoactive drug, S-carboxymethyl-L-cysteine (SCMC) and its thioether metabolites. Phenylalanine monooxygenase was shown to be able to catalyze the S-oxygenation of SCMC, its decarboxylated metabolite, S-methyl-L-cysteine and both their corresponding N-acetylated forms. However, thiodiglycolic acid was found not to be a substrate. The enzyme profiles for both phenylalanine and SCMC showed Michaelis-Menten with noncompetitive substrate inhibition for both the substrate-activated and the lysophosphatidylcholine-activated mPAH assays. The tetrameric enzyme was shown to undergo posttranslational activation by preincubation with substrate, lysophosphatidylcholine, N-ethylmaleimide (a thiol alkylating agent), and the proteolytic enzymes alpha-chymotrypsin and trypsin. Similar posttranslational activation of PAH activity in the rat and human has also been reported. These results suggest that in the mouse, PAH was responsible for the S-oxidation of SCMC and that the mouse models of the hyperphenylalaninemias may be a potential tool in the investigation of the S-oxidation polymorphism in man.

The activity of wild type and mutant phenylalanine hydroxylase with respect to the C-oxidation of phenylalanine and the S-oxidation of S-carboxymethyl- l-cysteine

The involvement of the enzyme, phenylalanine hydroxylase (PAH), in the S-oxidation of S-carboxymethyl- l-cysteine (SCMC) is now firmly established in man and rat. However, the underlying role of the molecular genetics of PAH in dictating and influencing the S-oxidation polymorphism of SCMC metabolism is as yet unknown. In this work we report that the S-oxidation of SCMC was dramatically reduced in the tetrahydrobiopterin (BH 4) responsive mutant PAH proteins (I65T, R68S, R261Q, V388M and Y414C) with these enzymes possessing between 1.2% and 2.0% of the wild type PAH activity when SCMC was used as substrate. These same mutant proteins express between 23% and 76% of the wild type PAH activity when phenylalanine was used as the substrate. The PAH mutant proteins (R158Q, I174T and R408W) that result in the classical phenylketonuria (PKU) phenotype expressing 0.2–1.8% of the wild type PAH activity when using phenylalanine as substrate were found to have <0.1% of the wild type PAH activity when SCMC was used as the substrate. Mutations that result in PAH proteins retaining some residual PAH activity with phenylalanine as substrate have <2.0% residual activity when SCMC was used as a substrate. This investigation has led to the hypothesis that the S-oxidation polymorphism in man is a consequence of an individual carrying one mutant PAH allele which has resulted in the loss of the ability of the residual PAH protein to undertake the S-oxidation of SCMC in vivo.

S‐CARBOXYMETHYLCYSTEINE INHIBITS CARBACHOL‐INDUCED CONSTRICTION OF EPITHELIUM‐DENUDED RAT AND HUMAN AIRWAY PREPARATIONS

1. The effects of s-carboxymethyl-L-cysteine (S-CMC), either administered orally to rats or incubated with tissue preparations from rats and humans, on isometric contractions of tracheal smooth muscle were investigated in the present study using an improved in vitro model of tracheal tube or ring preparations. The involvement of the tracheal epithelium in the observed effects was also investigated. 2. The experimental model permitted selective perfusion of the airway tube, luminal-IN or serosal-OUT, and measurement of airway smooth muscle contraction or relaxation in preparations with (+) or without (-) epithelium (Ep), excluding direct effects of airway mucus. 3. We found that oral pretreatment of rats with S-CMC (mixed with water; 200 mg/kg per day for 2 weeks), but not short pre-incubation of preparations in vitro (10(-3) mol/L S-CMC for 1 h), diminished the sensitivity of -Ep preparations to carbachol compared with controls (EC(50) (-log(10) mol/L) values: 5.5 +/- 0.1 vs 5.8 +/- 0.1, respectively, for IN perfusion (P < 0.005); 5.6 +/- 0.1 vs 5.9 +/- 0.1, respectively, for OUT perfusion (P < 0.005)), whereas the sensitivity of preparations to aminophylline was not affected. Normal sensitivity to carbachol stimulation was re-established if preparations were pre-incubated with capsaicin. 4. It was also found that longer pre-incubation (4 h) of ring-preparations of human bronchus with S-CMC (10(-5) mol/L) in vitro resulted in a diminished response to carbachol stimulation. 5. In conclusion, S-CMC had small inhibitory effects on the sensitivity of rat and human airway smooth muscle to carbachol, particularly in endothelium-denuded preparations. Whether the epithelium was responding to S-CMC by producing some contracting factor(s) requires further investigation.

Thiodiglycolic acid as a possible causative agent of fixed drug eruption provoked only after continuous administration of S-carboxymethyl-l-cysteine: case report and review of reported cases

Conflict of interest: None declared Sir, A provocation test of a fixed drug eruption (FDE) usually induces an erythematous response at the previously affected sites following a single, normal dose and the response is typically observed within 24 h.1, 2 We recently experienced two cases of FDE induced by S‐carboxymethyl‐L‐cysteine (SCMC) and found that in both cases it required the intake of the agent for more than two consecutive days to elicit the eruption. Similar observations have been described in the Japanese literature, although the reason is so far unknown.3, 4 We therefore characterized this feature of SCMC‐induced FDE and we discuss its possible aetiology. Two Japanese females (patient 1: 32 years old, patient 2: 8 years old) who had histories of a fixed drug eruption two days after consecutive ingestions of SCMC, were examined by oral challenge tests and patch tests. Oral challenge tests were performed with the consent of the patients and the parents. In both cases, skin eruptions were not induced by a single challenge of the usual dose of SCMC when it was given either in the morning or in the evening and the skin observed for 1 week. However, if SCMC was consecutively taken at night and three times on the following day, erythema developed by the morning of the third day. We performed patch tests with SCMC and its metabolites, S‐methyl‐L‐cysteine (SMC) (Sigma Chemical Co, Poole, Dorset, U.K.) and thiodiglycolic acid (TDA) (Sigma Chemical Co.). Both 1% and 10% concentrations of TDA showed positive patch tests at the affected skin sites in both patients. Neither SCMC nor SMC elicited any positive findings. Five healthy volunteers did not show a positive response to any of the three chemicals. Lymphocyte transformation tests for SCMC, SMC and TDA were negative in both patients.

Phenylalanine hydroxylase: possible involvement in the S-oxidation of S-carboxymethyl-l-cysteine

Activated phenylalanine 4-monooxygenase, phenylalanine hydroxylase (PAH), is known to be involved in the S-oxidation of a number of sulfide compounds. One of these compounds, S-carboxymethyl-l-cysteine (SCMC), is currently used for the treatment of chronic obstructive pulmonary disease and otitis media with effusion as a mucolytic agent, and the S-oxides are the major metabolites found in urine. However, the enzyme catalyzing the S-oxidation of SCMC has yet to be identified. Here we report on the role of nonactivated phenylalanine 4-monooxygenase activity in rat liver cytosol in the S-oxidation of SCMC. Linearity of the enzyme assays was seen for both time (0–16min) and cytosolic protein concentration (0.1–0.5mg/ml). The calculated Km and Vmax values for the formation of SCMC (S) S-oxide were 3.92±0.15mM and 1.10±0.12nmol SCMC (S) S-oxide formed/mg protein/min, respectively. The calculated Km and Vmax values for the formation of SCMC (R) S-oxide were 9.18±1.13mM and 0.46±0.11nmol SCMC (R) S-oxide formed/mg protein/min, respectively. These results indicate that in the female Wistar rat, nonactivated PAH showed a stereospecific preference for the formation of the (S) S-oxide metabolite of SCMC against the (R) S-oxide metabolite of SCMC.

Phenylalanine 4-Monooxygenase and the S-Oxidation of S-Carboxymethyl-L-cysteine

The identity of the enzyme responsible for the S-oxidation of the mucolytic S-substituted L-cysteine drug, S-carboxymethyl-L-cysteine (SCMC), has been actively investigated for the last 10 years. A genetic polymorphism exists in the oxidation of the thioether moiety that has been identified as a disease susceptibility factor in a number of degenerative diseases. This polymorphism has also been implicated in the wide variation in clinical response to SCMC therapy in man. To date little is known about the molecular enzymology of this reaction but a previous investigation revealed that rat activated phenylalanine 4-monooxygenase (PAH) could S-oxidise both Met- and S-methyl-L-cysteine (SMC) to their S-oxide metabolites. We have investigated the hypothesis that SCMC was also a substrate for activated PAH in the cytosolic faction of the Wistar rat. 1. Substrate and inhibitor investigation revealed that SCMC was a substrate for activated PAH activity in vitro. 2. The large aromatic amino acid hydroxylase monoclonal antibody and the Fe3+ chelator, deferoxamine, completely inhibited both Phe and SCMC oxidation to their respective metabolites. 3. Analysis of the Dixon plots revealed that both Phe and SCMC competitively inhibited each other's oxidation. 4. Correlation studies showed that the rate of production of Tyr was positively correlated to the production of both SCMC and SMC S-oxides in 20 female Wistar rat hepatic cytosolic fractions. These results strongly support the hypothesis that PAH is the enzyme responsible for SCMC S-oxidation in the rat.

Lack of congruence between cysteine dioxygenase activity and S-carboxymethyl-L-cysteine S-oxidation activity in rat cytosol.

The identity of the enzyme(s) responsible for the S-oxidation of the mucoactive drug S-carboxymethyl-L-cysteine (SCMC) is unknown but the protein(s) are a susceptibility factor for a number of chronic degenerative diseases. The structural similarities between the amino acid L-cysteine and SCMC have raised the possibility that cysteine dioxygenase (CDO) may be responsible for this biotransformation reaction. Both CDO and SCMC S-oxygenase were found to require Fe2+ for enzymatic activity, and both enzyme activities were inhibited by Fe2+ and Fe3+ chelators. However, sulphydryl group modification of the enzymes resulted in the activation of the S-oxidation of SCMC but inhibition of the S-oxidation of L-cysteine. When the two enzyme activities were quantified in 20 female hepatic cytosolic fractions no linear correlation in the production of their respective metabolites was seen. The results of this investigation indicate that CDO is not responsible for the S-oxidation of SCMC in the rat.

Protective effect of erdosteine against hypochlorous acid-induced acute lung injury and lipopolysaccharide-induced neutrophilic lung inflammation in mice.

The effect of erdosteine, a mucoactive drug, on hypochlorous acid (HOCl)-induced lung injury, and the lipopolysaccharide (LPS)-induced increase in tumour necrosis factor-alpha (TNF-alpha) production and neutrophil recruitment into the airway, was investigated. Male BALB/c mice were orally administered erdosteine (3-100 mgkg(-1)), ambroxol hydrochloride (ambroxol) (3-30 mgkg(-1)), S-carboxymethyl-L-cysteine (S-CMC) (100-600 mgkg(-1)) or prednisolone (10 mgkg(-1)), 1 h before intratracheal injection of HOCl or LPS. In the HOCl-injected mice, erdosteine markedly suppressed increases in the ratios of lung wet weight to bodyweight and lung dry weight to bodyweight, whereas the other mucoactive drugs ambroxol and S-CMC had little effect. Erdosteine also inhibited the LPS-induced neutrophil influx, although it did not affect the increased level of TNF-alpha in the bronchoalveolar lavage fluid. The results suggest that attenuation of reactive oxygen species and neutrophil recruitment is involved in the clinical efficacy of erdosteine in the treatment of chronic bronchitis.

Phenotypic variation in xenobiotic metabolism and adverse environmental response: focus on sulfur-dependent detoxification pathways

Proper bodily response to environmental toxicants presumably requires proper function of the xenobiotic (foreign chemical) detoxification pathways. Links between phenotypic variations in xenobiotic metabolism and adverse environmental response have long been sought. Metabolism of the drug S-carboxymethyl- l-cysteine (SCMC) is polymorphous in the population, having a bimodal distribution of metabolites, 2.5% of the general population are thought to be nonmetabolizers. The researchers developing this data feel this implies a polymorphism in sulfoxidation of the amino acid cysteine to sulfate. While this interpretation is somewhat controversial, these metabolic differences reflected may have significant effects. Additionally, a significant number of individuals with environmental intolerance or chronic disease have impaired sulfation of phenolic xenobiotics. This impairment is demonstrated with the probe drug acetaminophen and is presumably due to starvation of the sulfotransferases for sulfate substrate. Reduced metabolism of SCMC has been found with increased frequency in individuals with several degenerative neurological and immunological conditions and drug intolerances, including Alzheimer's disease, Parkinson's disease, motor neuron disease, rheumatoid arthritis, and delayed food sensitivity. Impaired sulfation has been found in many of these conditions, and preliminary data suggests that it may be important in multiple chemical sensitivities and diet responsive autism. In addition, impaired sulfation may be relevant to intolerance of phenol, tyramine, and phenylic food constituents, and it may be a factor in the success of the Feingold diet. These studies indicate the need for the development of genetic and functional tests of xenobiotic metabolism as tools for further research in epidemiology and risk assessment.

Chelating extraction of lead and copper from an authentic contaminated soil using N-(2-acetamido)iminodiacetic acid and S-carboxymethyl- l-cysteine

Heavy metal contamination of soil is common at many hazardous waste sites in many industrialized nations. Lead, chromium, cadmium, copper, zinc, and mercury are most often encountered at elevated concentrations. The extraction of heavy metals using selected chelating agents is seen as a remediation technique. In this study, we examined the chelating extraction and recovery of lead and copper from an authentic contaminated soil using S-carboxymethyl- l-cysteine (SCMC) and N-(2-acetamido)iminodiacetic acid (ADA) as a function of contact time, pH, ligand concentration, soil suspension, and number of consecutive runs. The results suggested that the chelators effectively extracted lead and cooper from the contaminated soil, with ADA particularly effective for lead, the major contaminant of the soil. The chelators were recovered and reused over consecutive runs with no loss in performance. The extraction effectiveness depended on chelator concentration but not on pH or solid contents of the suspension. Major findings from this study of an authentic soil were consistent with our previous efforts in the extraction of heavy metals from spiked soils.

Quantitative investigation of copper(II) and zinc(II) complexes with S-carboxymethyl-L-cysteine and computer-simulated appraisal of their potential significance in vivo

S-carboxymethyl- l-cysteine (SCC) is a mucolytic agent extensively used in the treatment of respiratory tract disorders. Some of the undesirable side effects observed during SCC therapy being reminiscent of symptoms characteristic of copper and zinc imbalances, the objective of this paper was to test the possible interference of SCC with the metabolism of these two metals. Copper(II)- and zinc(II)-SCC complex equilibria have thus been investigated under physiological conditions by means of classical potentiometry combined with computer-assisted calculation techniques. Formation constants derived from these studies have then been used to simulate 1) the potential influence of SCC on the distribution of the above metals in blood plasma and 2) the extent to which gastrointestinal interactions between the drug and each metal ion in turn are likely to affect the bioavailability of each other. The results of these simulations show that 1) plasma therapeutic levels of SCC are not likely to induce dramatic changes in the distributions of copper(II) and zinc(II) low molecular weight fractions, 2) the gastrointestinal distribution of the drug is not affected by standard dietary doses of these metals, and 3) in contrast, therapeutic concentrations of SCC are capable of mobilizing significant fractions of both metals into tissue-diffusible electrically neutral complexes In conclusion significant depletions of neither corner nor zinc are to be expected from oral administration of SCC. While the drug may to some extent facilitate the excretion of Cu 2+ and Zn 2+ ions from blood plasma, its gastrointestinal influence is, on the contrary, favorable to a better absorption of these two metals.

Pharmacogenetics of the S-oxidation of S-carboxymethyl-L-cysteine.

The pharmacogenetics of S-carboxymethyl-L-cysteine (SCMC) have been studied in detail. When results from administration of SCMC to 200 volunteers were analysed, there was seen to be a wide interindividual variation in the percentage of sulphoxide metabolites excreted. Computer assisted analysis suggested that the population distribution observed could be most economically represented as two overlapping Gaussian distributions with the smaller mode representing poor sulphoxidisers. This phenomenon appears to be largely genetic in origin and to behave as though controlled by one autosomal recessive gene, but environmental factors may also be important. Poor sulphoxidisers seem to be overrepresented in certain patient populations with chronic diseases. These findings are discussed in terms of oxidative metabolism of sulphur-containing compounds.

An investigation of the metabolism of S-carboxymethyl-L-cysteine in man using a novel HPLC-ECD method.

The metabolism of an oral dose of S-carboxymethyl-L-cysteine (SCMC) in man has been studied; the quantitative determination of SCMC, S-methyl-L-cysteine (SMC) and their sulphoxide metabolites (SCMCO and SMCO), in urine, was carried out using high performance liquid chromatography (HPLC) with electrochemical detection (ECD); the possibility of stereospecific sulphoxidation was investigated.

Determination of the sulphoxide metabolites of S-carboxymethyl-L-cysteine by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method has been developed for the determination of the mucolytic drug S-carboxymethyl-L-cysteine (SCMC) and its major metabolites S-methyl-L-cysteine (SMC), SCMC-sulphoxide and SMC-sulphoxide. Pre-column derivatisation with o-phthalaldehyde ethanethiol and electrochemical detection were employed. The mobile phase was methanol-phosphate buffer of various compositions. SCMC and its sulphoxide were determined simultaneously but, for optimum resolution, they were determined separately using a different mobile phase composition for each. The method, which was convenient and rapid, allowed the resolution of the sulphoxide diastereoisomers. Good recoveries of SCMC and its metabolites were obtained from spiked urine samples. All four compounds were detected in the urine of human volunteers during a 24-h period following a single dose of the drug. The major constituent recovered was unchanged SCMC.

Cytosolic sulphoxidation of S-carboxymethyl-L-cysteine in mammals

Cytosolic sulphoxidation of S-carboxymethyl-L-cysteine in mammals

Polymorphic sulphoxidation of S-carboxymethyl-L-cysteine in man

Polymorphic sulphoxidation of S-carboxymethyl-L-cysteine in man

S-Carboxymethyl-L-cysteine

S-Carboxymethyl-L-cysteine

S-Carboxymethyl-L-cysteine sulfoxide (configuration 2R:4R)

S-Carboxymethyl-L-cysteine sulfoxide (configuration 2R:4R)