Efficacy of the oxime HI-6 dimethanesulphonate in the treatment of guinea-pigs exposed to the nerve agents GB and GD.

The bispyridinium oxime HI-6 DMS is in development as an improved therapy for the treatment of patients exposed to organophosphorus nerve agents. The aim of the work described in this paper was to provide non-clinical data to support regulatory approval of HI-6 DMS, by demonstrating efficacy against an oxime-sensitive agent, GB and an oxime-resistant agent, GD. We investigated the dose-dependent protection afforded by therapy including atropine, avizafone and HI-6 DMS in guinea-pigs challenged with GB or GD. We also compared the efficacy of 30 mg.kg-1 of HI-6 DMS to an equimolar dose of the current in-service oxime P2S and the dichloride salt of HI-6 (HI-6 Cl2). In the treatment of GB or GD poisoning there was no significant difference between the salt forms. The most effective dose of HI-6 DMS in preventing lethality following challenge with GB was 100 mg.kg-1; though protection ratios of at least 25 were obtained at 10 mg.kg-1. Protection against GD was lower, and there was no significant increase in effectiveness of HI-6 DMS doses of 30 or 100 mg.kg-1. For GD, the outcome was improved by the addition of pyridostigmine pre-treatment. These data demonstrate the benefits of HI-6 DMS as a component of nerve agent therapy. © Crown copyright (2023), Dstl.

S-Carboxymethyl-l-cysteine: a multiple dosing study using pharmacokinetic modelling.

S-Carboxymethyl-l-cysteine is a mucolytic agent used as adjunctive therapy in the treatment of respiratory disorders. Various mechanisms of action have been proposed but few studies have attempted to link the required in vitro concentrations with those achieved actually in vivo during clinical therapy.The data from several published studies has been re-analysed by WinNonlin using non-compartmental analysis modelling, Phoenix modelling and Classic PK compartmental modelling for both single (500-1500 mg) and multiple oral administration of the drug.Multiple dose modelling indicated maximum peak concentrations (Cmax) ranging from 1.29 to 11.22 µg/ml and those at steady state (Css(av)) from 1.30 to 8.40 µg/ml. For the standard therapeutic regimen of 3 × 750 mg (2250 mg/day) these values were 1.29-5.22 µg/ml (Cmax) and 1.30-3.50 µg/ml (Css(av)). No accumulation was observed.Hence, only the pharmacodynamic studies reporting significant effects below c.10 µg/ml were likely to occur in vivo and these were mainly gene-related mechanisms. The majority of events, although demonstrable in vitro, required levels much greater than possible to achieve in the clinical situation.Such unappreciated disregard for in vitro-in vivo 'concentration matching' may lead to erroneous conclusions regarding mechanisms of action for many drugs as well as for S-carboxymethyl-l-cysteine.

Phenylalanine 4-monooxygenase: the "sulfoxidation polymorphism".

1. Consistent differences in the proportion of an orally administered dose of S-carboxymethyl-l-cysteine subsequently excreted in the urine as S-oxide metabolites were reported 40 years ago. This observation suggested the existence of inter-individual variation in the ability to undertake the enzymatic S-oxygenation of this compound. Pedigree studies and investigations employing twin pairs indicated a genetically controlled phenomenon overlaid with environmental influences. It was reproducible and not related to gender or age.2. Studies undertaken in several healthy volunteer cohorts always provided similar results that were not significantly different when statistically analysed. However, when compared to these healthy populations, a preponderance of subjects exhibiting the characteristic of poor sulfoxidation of S-carboxymethyl-l-cysteine was found within groups of patients suffering from various disease conditions. The most striking of these associations were witnessed amongst subjects diagnosed with neurodegenerative disorders; although, underlying mechanisms were unknown.3. Exhaustive investigation has identified the enzyme responsible for this S-oxygenation reaction as the tetrahydrobiopterin-dependent aromatic amino acid hydroxylase, phenylalanine 4-monooxygenase classically assigned the sole function of converting phenylalanine to tyrosine. The underlying principle is discussed that enzymes traditionally associated solely with intermediary metabolism may have as yet unrecognised alternative roles in protecting the organism from potential toxic assault.

Drug S-oxidation and phenylalanine hydroxylase: a biomarker for neurodegenerative susceptibility in Parkinson's disease and amyotrophic lateral sclerosis.

Background The S-oxidation of S-carboxymethyl-L-cysteine has been reported previously to be a biomarker of disease susceptibility in Parkinson's disease and amyotrophic lateral sclerosis. In the present investigation, the original observations have been extended and confirmed. Methods Meta-analysis of previously published investigations into the S-oxidation polymorphism together with new subject data was evaluated. Results The incidence of the poor metaboliser phenotype (no urinary recovery of S-oxide metabolites) was found to be 3%-7% within healthy and non-neurological disease populations, whereas 38% of the Parkinson's disease subjects and 39% of the amyotrophic lateral sclerosis group were phenotyped as poor metabolisers. The consequent odds risk ratio of developing Parkinson's disease was calculated to be 33.8 [95% confidence interval (CI), 13.3-86.1] and for amyotrophic lateral sclerosis was 35.2 (95% CI, 13.0-85.1). Conclusions The possible involvement of the enzyme responsible for this S-oxidation biotransformation reaction, phenylalanine hydroxylase, should be further investigated to elucidate its potential role in the mechanism(s) of toxicity in susceptible individuals displaying these diseases. The "Janus hypothesis," possibly explaining why phenylalanine hydroxylase is a biomarker of neurodegenerative disease susceptibility, together with the general theme that this concept may apply to many other hitherto unsuspected enzyme systems, is presented.

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.

Xenobiotic Conjugation with Dicarboxylic Acids.

BACKGROUND: Although it is believed widely that the various routes of xenobiotic metabolism are now all known and effectively understood, occasionally there emerges a metabolite that signals a novel biotransformation pathway, especially where the xenobiotic may in some way interact with the myriad processes of intermediary metabolism. There are a few reports in the literature where saturated short-chain dicarboxylic acids have been exploited as conjugating agents and these unusual xenobiotic metabolites subsequently excreted intact in the urine. METHOD: Initially suggested by unpublished observations bolstered by extensive experience of the authors and colleagues in the field of xenobiochemistry, this narrative review has been supplemented by a search of bibliographic databases and the subsequent scrutiny of numerous peer-reviewed research articles. The resultant sparse and widely dispersed information has been examined, analysed and presented in this review. RESULTS: Xenobiotic conjugation with dicarboxylic acids has been demonstrated to occur within several domains of life; microorganisms, plants, invertebrates and mammals. However, considering the number of xenobiotic metabolism investigations that have been undertaken reports of such conjugations are exceedingly rare. CONCLUSION: Dicarboxylic acid condensation with xenobiotic molecules may occur at nitrogen centres, or more precisely with a primary or secondary amine, that is at nitrogen still possessing a replaceable hydrogen atom. Both aliphatic amines and arylamines may be substrates with many of the free amino groups being formed by previous Ndealkylation reactions. Hopefully, awareness of this metabolic route will be raised and researchers will be enthused to search for this type of conjugate.

Phenylalanine hydroxylase: A biomarker of disease susceptibility in Parkinson's disease and Amyotrophic lateral sclerosis.

The S-oxidation of S-carboxymethyl-l-cysteine has been reported previously to be a biomarker of disease susceptibility in Parkinson's disease and Amyotrophic lateral sclerosis. In this investigation, the original observations have been confirmed with the incidence of the poor metaboliser phenotype (no urinary recovery of S-oxide metabolites) being found to be 3.9% within healthy control population. However, 38.3% of the Parkinson's disease subjects and 39.0% of the Amyotrophic lateral sclerosis group were phenotyped as poor metabolisers. The consequent odds risk ratio of developing Parkinson's disease was calculated to be 15.5 (95% CI 9.5-25.3) and for Amyotrophic lateral sclerosis was 15.2 (95% CI 8.8-26.5). Thus, the possible role of the enzyme responsible for the S-oxidation biotransformation reaction, phenylalanine hydroxylase, must be further investigated to elucidate the mechanism(s) of toxicity in susceptible individuals displaying these diseases. A dual role potentially explaining of the role of phenylalanine hydroxylase as a biomarker of disease susceptibility is presented together with the observation that metabolomics is a possible way forward in the identification of potential pro-toxins/toxins in those individuals phenotyped as poor metabolisers (Controls, Parkinson's disease and Amyotrophic lateral sclerosis subjects).

Comparison of the sulfur-oxygenation of cysteine and S-carboxymethyl-l-cysteine in human hepatic cytosol and the rôle of cysteine dioxygenase.

OBJECTIVES: To determine the Km , Vmax , cofactor, activator and inhibitor requirements of human cysteine dioxygenase and S-carboxymethyl-l-cysteine S-oxygenase with respect to both l-Cysteine and S-carboxymethyl-l-cysteine as substrates. METHODS: In vitro human hepatic cytosolic fraction enzyme assays were optimised for cysteine dioxygenase activity using l-Cysteine as substrate and the effect of various cofactors, activators and inhibitors on the S-oxidations of both l-Cysteine and S-carboxymethyl-l-cysteine were investigated. KEY FINDINGS: The results of the in vitro reaction phenotyping investigation found that although both cysteine dioxygenase and S-carboxymethyl-l-cysteine S-oxygenase required Fe2+ for catalytic activity both enzymes showed considerable divergence in cofactor, activator and inhibitor specificities. Cysteine dioxygenase has no cofactor but uses NAD+ and NADH(H+ ) as pharmacological chaperones and is not inhibited by S-carboxymethyl-l-cysteine. S-carboxymethyl-l-cysteine S-oxygenase requires tetrahydrobiopterin as a cofactor, is not activated by NAD+ and NADH(H+ ) but is activated by l-Cysteine. Additionally, the sulfydryl alkylating agent, N-ethylmaleimide, activated carboxymethyl-l-cysteine S-oxygenase but inhibited cysteine dioxygenase. CONCLUSIONS: Human hepatic cytosolic fraction cysteine dioxygenase activity is not responsible for the S-oxidation of the substituted cysteine, S-carboxymethyl-l-cysteine.

Phenylalanine monooxygenase and the sulfur oxygenation of S-carboxymethyl-L-cysteine in mice.

1. The extent of sulfoxidation of the drug, S-carboxymethyl-L-cysteine, has been shown to vary between individuals, with this phenomenon being mooted as a biomarker for certain disease states and susceptibilities. Studies in vitro have indicated that the enzyme responsible for this reaction was phenylalanine monooxygenase but to date no in vivo evidence exists to support this assumption. Using the mouse models of mild hyperphenylalaninamia (enu1 PAH variant) and classical phenylketonuria (enu2 PAH variant), the sulfur oxygenation of S-carboxymethyl-L-cysteine has been investigated. 2. Compared to the wild type (wt/wt) mice, both the heterozygous dominant (wt/enu1 and wt/enu2) mice and the homozygous recessive (enu1/enu1 and enu2/enu2) mice were shown to have significantly increased Cmax, AUC(0-180 min) and AUC(0-∞ min) values (15 - to 20-fold higher). These results were primarily attributable to the significantly reduced clearance of S-carboxymethyl-L-cysteine (13 - to 22-fold lower). 3. Only the wild type mice produced measurable quantities of the parent S-oxide metabolites. Those mice possessing one or more allelic variant showed no evidence of blood SCMC (R/S) S-oxides. These observations support the proposition that differences in phenylalanine hydroxylase activity underlie the variation in S-carboxymethyl-L-cysteine sulfoxidation and that no other enzyme is able to undertake this reaction.

S-carboxymethyl-L-cysteine and it (R/S)-S-oxides in beagle dog plasma and hepatic cytosol.

1. Incubation of beagle hepatic cytosol, under conditions promoting phenylalanine hydroxylase activity, led to the formation of the sulfoxide derivatives of S-carboxymethyl-L-cysteine, N-acetyl-S-carboxymethyl-L-cysteine, S-methyl-L-cysteine and N-acetyl-S-methyl-L-cysteine. Thiodiglycolic acid was not a substrate. Enzyme kinetic parameters (Km, Vmax) were derived indicating S-carboxymethyl-L-cysteine had the greatest clearance; no enantioselective preference was observed for this S-oxygenation reaction. 2. Following oral administration of S-carboxymethyl-L-cysteine to beagle dogs, the parent substance and its sulfoxide were the only compounds identified in the plasma. Pharmacokinetic data have been obtained indicating that the small amount of sulfoxide formed persisted within the body for longer than the parent material, but that the majority of the ingested dose remained in the administered sulfide form. 3. The sulfide moiety within the muco-regulatory drug, S-carboxymethyl-L-cysteine, is thought to be vital as it acts as a free radical scavenger, resulting in the inactive sulfoxide. Additional extensive enyzme-mediated sulfoxidation would decrease the amount of active sulfide available. In the dog this appears to not be an issue, signalling possible exploitation for therapeutic benefit in treating airway disease.

S-carboxymethyl-L-cysteine.

S-carboxymethyl-L-cysteine, the side-chain carboxymethyl derivative of the sulfur-containing amino acid, cysteine, has been known and available for almost 80 years. During this time, it has been put to a variety of uses, but it is within the field of respiratory medicine that, presently, it has found a clinical niche. Early studies indicated that this compound underwent a rather simplistic, predictable pattern of metabolism, whereas later investigations alluded to more subtle interactions with the pathways of intermediary metabolism, as may be expected for an amino acid derivative. In addition, suggestions of polymorphic influences and circadian rhythms within metabolic profiles have emerged. These latter factors may underlie the conflicting reports regarding the therapeutic efficacy of this compound: that it appears to work well in some patients, but has no measurable effects in others. The relevant literature pertaining to the fate of this compound within living systems has been reviewed and a comprehensive précis advanced. Hopefully, this article will serve as a vade mecum for those interested in S-carboxymethyl-L-cysteine and as a catalyst for future research.

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.

Recombinant heteromeric phenylalanine monooxygenase and the oxygenation of carbon and sulfur substrates.

OBJECTIVES: The aim of this investigation was to provide in-vitro enzyme kinetic data to support the hypothesis that the in-vivo heterozygous dominant phenotype for phenylalanine monooxygenase (hPAH) was responsible for the S-oxidation polymorphism in the metabolism of S-carboxymethyl-l-cysteine reported in humans. Using a dual-vector expression strategy for the co-production of wild-type and mutant human hPAH subunits we report for the first time the kinetic parameters (K(m) , V(max) , CL(E) ) for the C-oxidation of l-phenylalanine and the S-oxidation of S-carboxymethyl-l-cysteine in homomeric wild-type, heteromeric mutant and homomeric mutant hPAH proteins in vitro. METHODS: A PRO(TM) dual-vector bacterial expression system was used to produce the required hPAH proteins. Enzyme activity was determined by HPLC with fluorescence detection. KEY FINDINGS: The heteromeric hPAH proteins (I65T, R68S, R158Q, I174T, R261Q, V338M, R408W and Y414C) all showed significantly decreased V(max) and CL(E) values when compared to the homomeric wild-type hPAH enzyme. For both substrates, all calculated K(m) values were significantly higher than homomeric wild-type hPAH enzyme, with the exception of I65T, R68S and Y414C heteromeric hPAH proteins employing l-phenylalanine as substrate. CONCLUSIONS: The net outcome for the heteromeric mutant hPAH proteins was a decrease significantly more dramatic for S-carboxymethyl-l-cysteine S-oxidation (1.0-18.8% of homomeric wild-type hPAH activity) when compared to l-phenylalanine C-oxidation (25.9-52.9% of homomeric wild-type hPAH activity) as a substrate. Heteromeric hPAH enzyme may be related to the variation in S-carboxymethyl-l-cysteine S-oxidation capacity observed in humans.

The pharmacokinetics of orally administered S-carboxymethyl-L-cysteine in the dog, calf and sheep.

The aim of this study was to investigate the feasibility of employing S-carboxymethyl-L-cysteine as a treatment of chronic obstructive pulmonary disease in dogs. To this end the pharmacokinetic parameters of orally administered S-carboxymethyl-L-cysteine were determined in the dog, cow and sheep. Six healthy beagle dogs, six endogenous Greek sheep and four Holstein Fresian calves were orally dosed with 10 mg/kg body weight of S-carboxymethyl-L-cysteine. No significant differences in T(max) and T(1/2) were reported between the species. However, significantly higher AUC((0-last)), 21.56+/-6.67 microg h ml(-1) and AUC((0-infinity)), 21.63+/-6.68 microg h ml(-1) were seen in the dogs compared to the sheep and calves. The calculated V(D) was significantly higher in the sheep (10.4+/-2.7 L kg(-1)) and the calves (3.8+/-0.7 L kg(-1)) compared to the dogs (1.0+/-0.6 L kg(-1)). The rank order of increasing C(L) was sheep (3.4+/-2.7 L h(-1)kg(-1))>calves (2.7+/-0.4 L h(-1) kg(-1))>dogs (0.5+/-0.2 L h(-1)kg(-1)). The result for the dogs was significantly lower that the calculated C(L) for the sheep and calves. All these results indicate that the oral administration of S-carboxymethyl-L-cysteine may be useful during the therapeutic management of chronic obstructive pulmonary disease in dogs.

Phenylalanine 4-monooxygenase and the role of endobiotic metabolism enzymes in xenobiotic biotransformation.

Phenylalanine 4-monooxygenase is the key enzyme in the sulfoxidation of the thioether drug S-carboxymethyl-l-cysteine and its thioether metabolites, S-methyl-l-cysteine, N-acetyl-S-carboxymethyl-l-cysteine and N-acetyl-S-methyl-l-cysteine in humans, and a number of other mammalian species. The kinetics constants of the sulfoxidation reaction (K(m), V(max) and CL(E)) have been investigated in cytosolic fractions derived from rat and human liver, in cytosolic fractions of HepG2 cells and using both human and mouse cDNA expressed phenylalanine 4-monooxygenase. Differences in K(m), V(max) and CL(E) of S-carboxymethyl-l-cysteine have been seen in HepG2 cells and human and mouse cDNA expressed phenylalanine 4-monooxygenase when compared to both rat and human hepatic cytosolic fractions. The association of the genetic polymorphism in the sulfoxidation of S-carboxymethyl-l-cysteine is highlighted with particular reference to this biotransformation reaction as being a biomarker of disease susceptibility in Parkinson's, Alzheimer's and motor neurone diseases and in rheumatoid arthritis. The possible underlying molecular genetics of the sulfoxidation polymorphism is also discussed in relation to the known allelic frequencies of phenylalanine 4-monooxygenase. Finally, the new found role phenylalanine 4-monooxygenase plays in xenobiotic metabolism is discussed.

Use of human microsomes and deuterated substrates: an alternative approach for the identification of novel metabolites of ketamine by mass spectrometry.

In vitro biosynthesis using pooled human liver microsomes was applied to help identify in vivo metabolites of ketamine by liquid chromatography (LC)-tandem mass spectrometry. Microsomal synthesis produced dehydronorketamine, seven structural isomers of hydroxynorketamine, and at least five structural isomers of hydroxyketamine. To aid identification, stable isotopes of the metabolites were also produced from tetra-deuterated isotopes of ketamine or norketamine as substrates. Five metabolites (three hydroxynorketamine and two hydroxyketamine isomers) gave chromatographically resolved components with product ion spectra indicating the presence of a phenolic group, with phenolic metabolites being further substantiated by selective liquid-liquid extraction after adjustments to the pH. Two glucuronide conjugates of hydroxynorketamine were also identified. Analysis by LC-coupled ion cyclotron resonance mass spectrometry gave unique masses in accordance with the predicted elemental composition. The metabolites, including the phenols, were subsequently confirmed to be present in urine of subjects after oral ketamine administration, as facilitated by the addition of deuterated metabolites generated from the in vitro biosynthesis. To our knowledge, phenolic metabolites of ketamine, including an intact glucuronide conjugate, are here reported for the first time. The use of biologically synthesized deuterated material as an internal chromatographic and mass spectrometric marker is a viable approach to aid in the identification of metabolites. Metabolites that have particular diagnostic value can be selected as candidates for chemical synthesis of standards.

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.

Human phenylalanine monooxygenase and thioether metabolism.

OBJECTIVES: The substrate specificity of wild-type human phenylalanine monooxygenase (wt-hPAH) has been investigated with respect to the mucoactive drug, S-carboxymethyl-L-cysteine and its thioether metabolites. The ability of wt-hPAH to metabolise other S-substituted cysteines was also examined. METHODS: Direct assays of PAH activity were by HPLC with fluorescence detection; indirect assays involved following disappearance of the cofactor by UV spectroscopy. KEY FINDINGS: wt-hPAH catalysed the S-oxygenation of S-carboxymethyl-L-cysteine, its decarboxylated metabolite, S-methyl-L-cysteine, and both their corresponding N-acetylated forms. However, thiodiglycolic acid was not a substrate. The enzyme profiles for both phenylalanine and S-carboxymethyl-L-cysteine showed allosteric kinetics at low substrate concentrations, with Hill constants of 2.0 and 1.9, respectively, for the substrate-activated wt-hPAH. At higher concentrations, both compounds followed Michaelis-Menten kinetics, with non-competitive substrate inhibition profiles. The thioether compounds, S-ethyl-L-cysteine, S-propyl-L-cysteine and S-butyl-L-cysteine were all found to be substrates for phenylalanine monooxygenase. CONCLUSIONS: Phenylalanine monooxygenase may play a wider role outside intermediary metabolism in the biotransformation of dietary-derived substituted cysteines and other exogenous thioether compounds.