An investigation into possible xenobiotic-endobiotic inter-relationships involving the amino acid analogue drug, S-carboxymethyl-L-cysteine and plasma amino acids in humans.

The amino acid derivative, S-carboxymethyl-L-cysteine, is an anti-oxidant agent extensively employed as adjunctive therapy in the treatment of human pulmonary conditions. A major biotransformation route of this drug, which displays considerable variation in capacity in man, involves the oxidation of the sulfide moiety to the inactive S-oxide metabolite. Previous observations have indicated that fasted plasma L-cysteine concentrations and fasted plasma L-cysteine/free inorganic sulfate ratios were correlated with the degree of sulfoxidation of this drug and that these particular parameters may be used as endobiotic biomarkers for this xenobiotic metabolism. It has been proposed also that the enzyme, cysteine dioxygenase, was responsible for the drug sulfoxidation. Further in this theme, the degree of S-oxidation of S-carboxymethyl-L-cysteine in 100 human volunteers was investigated with respect to it potential correlation with fasted plasma amino acid concentrations. Extensive statistical analyses showed no significant associations or relationships between the degree of drug S-oxidation and fasted plasma amino acid concentrations, especially with respect to the sulfur-containing compounds, methionine, L-cysteine, L-cysteine sulfinic acid, taurine and free inorganic sulfate, also the derived ratios of L-cysteine/L-cysteine sulfinic acid and L-cysteine/free inorganic sulfate. It was concluded that plasma amino acid levels or derived ratios cannot be employed to predict the degree of S-oxidation of S-carboxymethyl-L-cysteine (or vice versa) and that it is doubtful if the enzyme, cysteine dioxygenase, has any involvement in the metabolism of this drug.

A comprehensive understanding of thioTEPA metabolism in the mouse using UPLC-ESI-QTOFMS-based metabolomics.

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.

An occupational hygiene investigation of exposure to acrylamide and the role for urinary S-carboxyethyl-cysteine (CEC) as a biological marker.

Acrylamide has a range of toxicological hazards including neurotoxicity and reproductive toxicity; however, occupational risk management is driven by its genotoxic and carcinogenic potential (it is classified within the EU as a Category 2 carcinogen, R45 and Category 2 mutagen, R46). Since there is the potential for skin absorption and systemic toxicity, biological monitoring may be a useful aid for the assessment of exposure via inhalation, ingestion and dermal absorption. However, there are currently no biological monitoring guidance values (BMGVs). This study describes an extensive survey of potential workplace exposure to acrylamide at the Ciba (Bradford) site to gather data suitable for a BMGV. This manufacturing site is typical within the industry as a whole and includes a cross section of activities and tasks representative of acrylamide exposure. Acrylamide is used in the manufacture of polyacrylamide based products for applications in water treatment; oil and mineral extraction; paper, paint and textile processes. Workers (62 plus 6 controls) with varying potential exposures provided a total of 275 pre shift and 247 post-shift urine samples along with 260 personal air samples. A small non-exposed control group was similarly monitored. Urine samples were analysed for S-carboxyethyl-cysteine (CEC). Airborne, surface and glove samples were analysed for acrylamide. Inhalation exposures were well controlled with values consistently below one-tenth of the UK Workplace Exposure Limit. Engineering controls, personal protective equipment and work practice, all contributed to good control of occupational exposure. CEC was found in urine samples from both exposed workers and non-occupationally exposed controls. At the low levels of exposure found, smoking made a significant contribution to urinary CEC levels. Nevertheless a correlation between urinary CEC and airborne acrylamide was found. A mixed effects model incorporating inhalation concentrations of acrylamide and smoking habits could predict some of the variation in observed post-shift urine results but could be improved through the use of additional surface contamination data. However, the data does not suggest that dermal absorption was a major contributor to the systemic dose. Based on the 90th percentile of the data, inclusive of the effects of smoking and environmental factors, a value of 4 mmol mol(-1) creatinine is proposed as a pragmatic BMGV associated with good occupational hygiene practice and control of workplace exposure. CEC in urine analysis has the utility for routine use as a means to estimate biological uptake where there is a potential for significant exposure or loss of workplace control.

Degradation to sulphate of S-methyl-L-cysteine sulphoxide and S-carboxymethyl-L-cysteine sulphoxide in man.

A nearly complete recovery of radioactivity was achieved over 14 days following the oral administration of [35S]-S-methyl-L-cysteine sulphoxide and [35S]-S-carboxymethyl-L-cysteine sulphoxide to four healthy male volunteers. The urine was the major pathway of excretion of radioactivity (c. 96% in 0-14 days; c. 59% in 0-24 hours), with the faecal route being relatively unimportant (c. 1.7% in 0-3 days). Inorganic sulphate was an important degradation product, incorporating a substantial proportion of radioactive sulphur derived from these molecules (c. 40% in 0-14 days; c. 20% in 0-24 hours). Subtle differences were noted in the pattern of radioactive sulphate excretion following administration of the two cysteine-sulphoxide compounds, suggesting that their sulphur-containing moieties may enter different catabolic routes.

Diurnal variation in the metabolism of S-carboxymethyl-L-cysteine in humans.

The routes of metabolism of S-carboxymethyl-L-cysteine in humans are dependent on the time of dosing. Administration of 750 mg of S-carboxymethyl-L-cysteine (Day 1) during the day at 8:00 AM followed by a 8:00 AM to 4:00 PM urine collection revealed that S-carboxymethyl-L-cysteine S-oxide was the major urinary metabolite produced. The 4:00 PM to midnight urine collection resulted in S-(carboxymethylthio)-L-cysteine being identified as the major urinary metabolite. However, the administration of 750 mg of S-carboxymethyl-L-cysteine (day 15) during the night at midnight and analysis of the midnight to 8:00 AM urine collection found that thiodiglycolic acid was the major urinary metabolite, whereas thiodiglycolic S-oxide was identified as the major urinary metabolite in the 8:00 AM to 4:00 PM urine collection. A diurnal variation in the metabolism of S-carboxymethyl-L-cysteine was seen and, in particular, the timing of S-carboxymethyl-L-cysteine administration had a profound effect on the identity of urinary S-oxide metabolites produced. After administration at 8:00 AM the urinary S-oxides produced were S-carboxymethyl-L-cysteine S-oxide and S-methyl-L-cysteine S-oxide but at midnight the major urinary S-oxide metabolite produced was thiodiglycolic acid S-oxide.

Re-evaluation of the metabolism of carbocisteine in a British white population.

It has been claimed that the amino acid derivative carbocisteine is predominantly metabolized by sulfoxidation and that this pathway exhibits a genetic polymorphism. Moreover, those subjects with a 'poor metabolizer' phenotype have been thought to have a genetic predisposition to developing certain diseases. We have confirmed the observations of others that this marker drug does not undergo significant S-oxidation. Furthermore, a novel urinary metabolite, S-(carboxymethylthio)-L-cysteine (CMTC) has recently been identified. To determine if a genetic polymorphism for this biotransformation pathway exists, metabolic ratios (% urinary excretion carbocisteine/% urinary excretion CMTC) for 120 healthy volunteers were assessed using high-performance thin-layer chromatography. Urinary excretion of the parent drug ranged from 6% of the dose administered to 56% (mean +/- SD, 23.4 +/- 0.8%). No cysteinyl sulfoxide metabolites were identified in the urine samples. The amount excreted as CMTC exhibited a 12-fold variation but only accounted for mean of 4.4% (1-12%) of the dose given. Two individuals initially had high metabolic ratios (> 30), however, on rechallenge both their MRs were less than 5. Therefore, carbocisteine is not an appropriate probe drug for sulfoxidation. The formation of the novel metabolite CMTC appears to exhibit polymorphism, although the considerable intra-subject variation for its formation does not allow assignment of a phenotype.

Determination of S-carboxymethyl-L-cysteine and some of its metabolites in urine and serum by high-performance liquid chromatography using fluorescent pre-column labelling.

Pre-column labelling techniques are described for the determination of S-carboxymethyl-L-cysteine (CMC) and its metabolites in urine and plasma samples by high-performance liquid chromatography (HPLC) without prior extraction. All substances containing an amino group were converted into fluorescent fluorenylmethyl derivatives with 9-fluorenylmethyloxycarbonyl chloride (FMOC). Deaminated or N-acetylated carbocysteine metabolites were coupled with 1-pyrenyldiazomethane (PDAM) to give fluorescent PDAM esters. Similar results were obtained with the two commercially available and stable diazomethane derivatives PDAM and 9-anthryldiazomethane (ADAM). Following double derivatization with PDAM and FMOC, in a single chromatographic run with two fluorescence detectors connected in series, amines and amino(carboxylic) acids could be detected by their FMOC residues and, simultaneously, carboxylic acids were detected as fluorescent PDAM esters. The (R) and (S) enantiomers of the sulphoxides of CMC, of methylcysteine and of N-acetyl CMC were separated, although the reversed-phase HPLC system did not contain a chiral additive or stationary phase designed for the separation of enantiomers. The methods do not include liquid extraction steps and can therefore be performed either manually or automatically using an HPLC autosampler. These methods were used for the investigation of a disputed pharmacogenetic polymorphism of S-oxidation of CMC in humans, which until now has most often been studied using paper chromatography. The described techniques were applied to the determination of CMC and its metabolites in human urine and plasma samples.

Identification of thiodiglycolic acid, thiodiglycolic acid sulfoxide, and (3-carboxymethylthio)lactic acid as major human biotransformation products of S-carboxymethyl-L-cysteine.

S-Carboxymethyl-L-cysteine (CMC) is used both as an orally administered mucolytic agent and as a probe drug for uncovering polymorphic sulfoxidation of other sulfur-containing drugs in humans. However, several recent studies could not confirm the formation of significant amounts of urinary sulfoxides of CMC or its decarboxylation product S-methyl-L-cysteine. The metabolism of CMC and a 13C-labeled isotopomer was therefore reinvestigated in 11 and 14 humans, respectively, and emphasis was laid on monitoring of potential alternative metabolic pathways. Combined capillary gas chromatography/electron impact or negative-ion chemical ionization mass spectrometry employing stable isotope-labeled analogues as internal standards were used for identification and quantification of CMC metabolites in human urine. Three nitrogen-free metabolites that were identified as thiodiglycolic acid (TDGA, mean: 19.8% of the dose/24 hr), thiodiglycolic acid sulfoxide (TDGA-SO, mean: 13.3% of the dose/24 hr), and (3-carboxymethylthio)lactic acid (TLA, mean: 2.1% of the dose/8 hr), cumulatively account for about one-third of the dose during a urinary collection period of 24 hr. In addition, trace amounts of both TDGA and TLA exist as endogenous components in urine from persons not administered exogenous CMC at levels of about 5 and 1 nmol/ml, respectively. Both major metabolites TDGA and TDGA-SO, that were not considered in previous sulfoxidation phenotyping, are predominantly excreted after 8 hr. These results demonstrate the existence of a pyruvate-like metabolic pathway and suggest the necessity of a revision of the hitherto accepted biotransformation route of CMC in humans.

Tracing the human metabolism of stable isotope-labelled drugs by ex vivo NMR spectroscopy. A revision of S-carboxymethyl-L-cysteine biotransformation.

A direct structural identification, and quantitative assessment below the 50 nmol/ml level, of the full pattern of renally excreted metabolites is made possible by 13C NMR measurements of untreated urine samples when stable isotope-labelled (13C) drug analogues are administered to humans. The full potential of the new ex vivo NMR approach is exemplified by a study, for a group of volunteers, of S-carboxymethyl-L-cysteine metabolism. The metabolic sulphoxidation pathway of S-carboxymethyl-L-cysteine in man, accepted so far, needs to be profoundly revised on the basis of the 13C NMR results.

Capillary electrophoretic determination of S-carboxymethyl-L-cysteine and its major metabolites in human urine: feasibility investigation using on-column detection of non-derivatized solutes in capillaries with minimal electroosmosis.

The capillary electrophoretic analysis of S-carboxymethyl-L-cysteine and some of its metabolites in human urine is reported using (i) on-column detection of underivatized solutes, (ii) minimal sample pretreatment and (iii) capillary columns with minimized electroosmosis. Experimental results obtained with two apparatus, the HPE-100 (Bio-Rad), featuring coated fused silica capillaries of 25 microns i.d., and with the Tachophor 2127 (LKB), having Teflon capillaries of 500 microns i.d. are discussed. Drug concentrations down to 0.2 mg/mL on capillary isotachophoresis and 0.03 mg/mL on capillary zone electrophoresis can be monitored with these instruments.

Poor sulphoxidation ability in patients with food sensitivity.

Patients with well defined reactions to foods were examined for their ability to carry out both sulphur and carbon oxidation reactions by using carbocisteine and debrisoquine as probe compounds. The proportion of poor sulphoxidisers (58 of 74) was significantly greater than that of a previously determined normal control population (67 of 200; p less than 0.005). The proportion of poor carbon oxidisers was not significantly different from the controls. Metabolic defects may play a part in the pathogenesis of adverse reactions to foods.

High pressure liquid chromatographic determination of carbocisteine in human plasma and urine.

A method is described for the direct analysis of the amino acid carbocisteine in plasma and urine samples, following reaction with dabsyl chloride. Dabsylated carbocisteine is subjected to high pressure liquid chromatography with spectrophotometric detection at 425 nm. The usefulness of the method for bioavailability studies is discussed and compared with methods currently in use.

The metabolism of S-carboxymethyl-L-cysteine in man: isolation of an ester glucuronic acid conjugate from urine.

A conjugate isolated from urine of human volunteers after an oral dose of S-carboxymethyl-L-cysteine was characterized as a carboxylic acid ester glucuronide. Of the 166 volunteers investigated, 61 gave no detectable drug glucuronide (less than 0.5% administered dose); the remaining 105 showed a unimodal distribution of drug glucuronide excretion accounting for 0.5-11.5% (mean 4.1%, median 3.4%) of the total dose recovered in the 0-8 h urine. No significant variation in subject age, sex or urinary pH was observed between the two groups, but those not excreting urinary glucuronide had significantly higher 0-8 h urine volumes (P less than 0.001), although notable exceptions occurred.

Disposition and metabolism in 1-(2-chloroethyl)-3-(2',3',4'-tri-o-acetyl, ribopyranosyl)-1-nitrosourea in rats.

The antineoplastic activity in animals of 1-(2-chloroethyl)-3-(2',3',4'-tri-O-acetyl, ribopyranosyl)-1-nitrosourea (RPCNU) has been widely demonstrated. The present study deals with the disposition and the metabolism of three 14C-labeled species of RPCNU. The chemical plasma half-life of the drug was less than 5 min. Within the first min after injections, most of the radioactivity derived from ethyl-14C groups were recovered as volatile products. Among these, 2-chloroethanol was identified as a main component. Analysis of labeled species in urine after administration of [ethyl-14C]RPCNU showed that thiodiacetic acid and its sulfoxide were major metabolites of RPCNU (62% of the urinary radioactivity). Traces of N-acetylcarboxymethyl- and N-acetylhydroxyethylcysteine) were also detected. The only labeled species concentrating in particular tissues was that carrying the chloroethyl moiety. Uptake to high levels of [ethyl-14C]RPCNU did occur in liver, kidney, pancreas, thymus, and Harder's gland.

The metabolism and elimination of S-carboxymethyl-L-cysteine in man.

The metabolism of 14C- and 35S-labeled S-carboxymethylcysteine (SCMC) has been compared. The majority of the dose appeared in urine in the first 24 hr; no activity was detectable in feces. Use of 25S-labeled compound gave small amounts of inorganic 35S-sulfate, whereas 14C-labeled material was degraded to give some [14C]urea. Peak excretion of the parent compound occurred first after administration of SCMC; metabolites requiring one chemical modification for their formation were maximally excreted 1-4 hr after the dose and those involving more metabolic processes appeared later (4-8 hr).

Variation in human metabolism of S-carboxymethylcysteine.

The metabolism of an oral dose of S-carboxymethylcysteine (SCMC) has been studied in man; the principal urinary component after 24 h was the unchanged compound (29.4-83.7%, 20 subjects). Markers variation was found between individuals; SCMC sulphoxide, S-methylcysteine, N-acetyl-S-carboxymethylcysteine, N-acetyl-S-methylcysteine with the corresponding sulphoxides and dicarboxymethylsulphide were also found as metabolites.

Identification of S-(carboxymethyl)-L-cysteine and thiodiglycolic acid, urinary metabolites of 2,2'-bis-(chloroethyl)-ether in the rat.

S-(carboxymethyl)-L-cysteine (CMC) and thiodiglycollic acid (TGA) were identified by gas chromatographic-mass spectrometric measurements in the urine of rats after intraperitoneal injections of 2,2'-bis-(chloroethyl)-ether (BCEE). It is therefore probable that BCEE is O-dealkylated by a mixed-function oxidation. The hepatocarcinogenic effect of BCEE may be explained by the liberation of chloroacetaldehyde in vivo.

Molecular mechanism of 1,1-dichloroethylene toxicity: excreted metabolites reveal different pathways of reactive intermediates.

The excretion and biotransformation of [14C] 1,1-dichloroethylene (vinylidene chloride, VDC) after administration of a single oral dose has been investigated in female rats. Seventy-two hours after a dose of 0.5, 5.0, and 50.0 mg/kg, 1.26, 9.70, 16.47%, respectively, are exhaled as unchanged VDC, and 13.64, 11.35, 6.13% as 14CO2. The main pathway of elimination is through renal excretion with 43.55, 53.88, 42.11% of the administered radioactivity. Through the biliary system, 15.74, 14.54, 7.65% of the activity are eliminated. The isolation of the main metabolites of VDC from 24 h urine is accomplished through the combined application of solvent extraction, ion exchange chromatography and thin layer chromatography. Then gas chromatography and mass spectrometry are used for their identification. Three metabolites have been identified: thiodiglycolic acid, N-acetyl-S-(2-carboxymethyl)cysteine and methyl-thio-acetylaminoethanol. In addition, three smaller unidentified radioactive peaks have been found. Thiodiglycolic acid is the main metabolite in VDC metabolism. The simultaneous formation of an ethanolamine- and a cysteine-conjugation product points to different reaction pathways of the postulated intermediate reactive epoxide; ethanolamine probably originates from membrane lipids, which react with VDC-epoxide and/or its derivatives. This pathway could explain, in part, the parenchyma damaging effect of VDC.