Identification of thymol phase I metabolites in human urine by headspace sorptive extraction combined with thermal desorption and gas chromatography mass spectrometry.

Development of a novel highly sensitive headspace sorptive extraction (HSSE) method in combination with thermal desorption gas chromatography coupled to a mass spectrometer (TD-GC/MS) allowed the identification of thymol and several phase I metabolites in human urine. Combined with an enzymatic hydrolysis of glucuronated or sulphated phase II metabolites of thymol and of the respective phase I metabolites prior to analysis, even trace quantities of hitherto not detected thymol phase I metabolites could be identified in urine samples of test persons after oral administration of 50mg thymol. It was proven, that human metabolism leads to a hydroxylation of the aromatic ring as well as of the iso-propyl side chain. Hydroxylation of the iso-propyl group results in the formation of the rather unstable p-cymene-3,8-diol and the corresponding dehydration product p-cymene-3-ol-8-ene which could be clearly detected in human urine samples. Furthermore, the aromatic hydroxylation products p-cymene-2,5-diol, its oxidation product p-cymene-2,5-dione and p-cymene-2,3-diol were also unambiguously identified by comparison with synthesized reference compounds.

Efeitos do timol sobre os parâmetros urinários envolvidos na formação de cálculos / Effects of thymol on urinary parameters related to lithogenesis

INTRODUÇÃO: Em estudo anterior, demonstramos que a acidificação ou alcalinização de amostras de urina no momento de entrega do material ao laboratório em comparação a amostras coletadas com conservantes não alterou os resultados urinários de parâmetros relacionados à investigação metabólica de litíase renal como o oxalato (OxU), cálcio (CaU), magnésio (MgU), ácido úrico (AcUrU) e creatinina (CreatU), com exceção do citrato (CitU), cujo valor foi discretamente menor. OBJETIVO: Avaliar se a adição de timol, por meio de sua ação antibacteriana, é capaz de prevenir a redução do CitU em amostras acidificadas 24 horas após a coleta, em relação às pré-acidificadas, sem interferir na determinação dos outros parâmetros urinários. MÉTODOS: 40 voluntários sadios coletaram uma amostra isolada de urina que foi dividida em quatro alíquotas de 10 ml contendo timol (1 g/l). Na primeira, o conservante ácido (HCl 6 N, 20 ml/l) foi adicionado imediatamente após a coleta e na segunda, somente após 24 horas. Além do CitU, nessas amostras também foram determinados OxU, CaU e MgU. Na terceira e quarta alíquotas, um conservante alcalino (NaHCO3, 5g/l) foi adicionado imediatamente ou 24 horas após a coleta para determinação do AcUrU. RESULTADOS: Na presença de timol, não se observou variação significante do CitU entre as urinas pré ou pós-acidificadas (577 ± 490 mg/l vs. 575 ± 501 mg/l). Os valores dos demais parâmetros também não sofreram alteração. CONCLUSÃO: A adição prévia de timol às amostras de urina permite que todos os parâmetros urinários litogênicos possam ser determinados numa mesma amostra, reduzindo o custo e o desconforto de múltiplas coletas de urina de 24 horas.

INTRODUCTION: In a previous study, we demonstrated that acidification or alkalinization of urine samples upon delivery of the material to the laboratory in comparison with samples with preservatives did not alter the results of urinary parameters related to the metabolic investigation into renal lithiasis such as oxalate (OxU), calcium (CaU), magnesium (MgU), uric acid (AcUrU) and creatinine (CreatU), with the exception of citrate (CitU), whose value was slightly lower. OBJECTIVE: To evaluate if the addition of thymol, through its antibacterial effect, is able to prevent the reduction of CitU observed in samples acidified 24 hs after collection in comparison with pre-acidified ones without interfering in the determination of other urinary parameters. METHODS: Forty (40) healthy volunteers collected a single spot urine sample, which was divided into four aliquots of 10 ml containing thymol (1 g/l). In the first sample, the acid preservative (HCl6N, 20 ml/l) was added immediately after collection and in the second, only after 24hs. OxU, CaU, CitU and MgU were determined. In the third and fourth aliquots, an alkali preservative (NaHCO3,5 g/l) was added immediately or 24 hs after collection for AcUrU determination. RESULTS: In the presence of thymol, there was no significant variation in CitU values between pre-or post-acidified samples (577±490 mg/l vs. 575±501 mg/l). The values of other parameters also remained unchanged. CONCLUSION: The prior addition of thymol to urine samples allows the determination of all lithogenic urinary parameters in the same sample, reducing the cost and inconvenience of multiple 24-hour urine collections.

Cytochrome P450-catalysed arene-epoxidation of the bioactive tea tree oil ingredient p-cymene: indication for the formation of a reactive allergenic intermediate?

1. The cytochrome P450-mediated metabolism of the tea tree oil ingredient p-cymene (p-isopropyltoluene) was studied by the application of in vitro enzymatic assays using different recombinant human cytochrome P450 enzymes. 2. In total, four enzymatic products were identified by gas chromatography-mass spectrometry. The enzymatic products identified were: thymol (2-isopropyl-5-methylphenol), p-isopropylbenzyl alcohol, p,alpha,alpha-trimethylbenzyl alcohol, and p-isopropylbenzaldehyde. 3. The enzymatic products of p-cymene resulted from catalysed enzymatic arene-epoxidation and hydroxylation reactions by the studied cytochrome P450 enzymes. 4. An in vivo study could only confirm the formation of one enzymatic product, namely thymol. Thymol was identified after enzymatic hydrolysis of glucuronide and sulphate conjugates in collected blood and urine samples. 5. The obtained results may help to increase the understanding of cases where skin sensitization and irritation by tea tree oil-containing products that are involved with allergic reactions of users of these products. The results also indicate that skin sensitization and irritation reactions not only can be explained by the frequently in literature reported auto-oxidation of tea tree resulting in bioactive oxidized products, but also now by the formation of epoxide intermediates resulting from catalysed arene-epoxidation reactions by selected human cytochrome P450 enzymes which are also located in different organs in humans.

Systemic availability and pharmacokinetics of thymol in humans.

Essential oil compounds such as found in thyme extract are established for the therapy of chronic and acute bronchitis. Various pharmacodynamic activities for thyme extract and the essential thyme oil, respectively, have been demonstrated in vitro, but availability of these compounds in the respective target organs has not been proven. Thus, investigation of absorption, distribution, metabolism, and excretion are necessary to provide the link between in vitro effects and in vivo studies. To determine the systemic availability and the pharmacokinetics of thymol after oral application to humans, a clinical trial was carried out in 12 healthy volunteers. Each subject received a single dose of a Bronchipret TP tablet, which is equivalent to 1.08 mg thymol. No thymol could be detected in plasma or urine. However, the metabolites thymol sulfate and thymol glucuronide were found in urine and identified by LC-MS/MS. Plasma and urine samples were analyzed after enzymatic hydrolysis of the metabolites by headspace solid-phase microextraction prior to GC analysis and flame ionization detection. Thymol sulfate, but not thymol glucuronide, was detectable in plasma. Peak plasma concentrations were 93.1+/-24.5 ng ml(-1) and were reached after 2.0+/-0.8 hours. The mean terminal elimination half-life was 10.2 hours. Thymol sulfate was detectable up to 41 hours after administration. Urinary excretion could be followed over 24 hours. The amount of both thymol sulfate and glucuronide excreted in 24-hour urine was 16.2%+/-4.5% of the dose.

Metabolism in rats of p-cymene derivatives: carvacrol and thymol.

The metabolism of carvacrol and thymol in rats was studied using gas chromatographic-mass spectrometric methods. The urinary excretion of metabolites was rapid. Only very small amounts were excreted after 24 hrs. Although large quantities of carvacrol and, especially, thymol were excreted unchanged (or as their glucuronide and sulphate conjugates), extensive oxidation of the methyl and isopropyl groups also occurred. This resulted in the formation of derivatives of benzyl alcohol and 2-phenylpropanol and their corresponding carboxylic acids. In contrast, ring hydroxylation of the two phenols was a minor reaction.

On the metabolic detoxication of thymol in rabbit and man.

The metabolic detoxication of thymol was investigated in rabbit and man. Thymol glucuronide which the aglycone is intact, was isolated from thymol medicated rabbit urine and identified as a acetyl derivative of methyl glucuronate. The hydroxylated product of thymol, thymohydroquinone, was recognized in a small amount in thymol medicated human urine. It was presumed that thymohydroquinone is excreted as ethereal sulfuric acid conjugate in man.

On the metabolic detoxication of 4-iodothymol in man.

The detoxication of 4-iodothymol was investigated in man. A new metabolite was isolated as a crystalline derivative from 4-iodothymol medicated urine and identified as 4-iodothymol glucuronide as a methyl acetyl derivative. The deiodinated metabolite of 4-iodothymol, thymoquinone, was also recognized in a very small amount in 4-iodothymol medicated urine.