RESUMEN
A method was developed for the determination of the monoterpene alcohols verbenol, myrtenol, perillyl alcohol, alpha-terpineol, Delta(3)-carene-10-ol, thymol and p-alpha,alpha-trimethylbenzylalcohol in urine samples. After an enzymatic cleavage of their glucuronide- and sulfate conjugates the monoterpene alcohols were converted in the urine matrix with 7-diethylaminocoumarin-3-carbonylazide into monoterpene-[7-(diethylamino)-coumarin-3-yl]-carbamate derivates prior to analyses. Enrichment of the monoterpene alcohols from the urine matrix was achieved by online-solid phase extraction (SPE) with restricted-access material (RAM). After removal of excess derivatization reagent and urine matrix components, the monoterpene derivatives were separated by high-performance liquid chromatography (HPLC) in combination with fluorescence (FLD) detection and simultaneous mass spectrometric (MS) identification. Detection limits (LOD) for studied monoterpene alcohols ranged between 22 and 197 ng/L. The method was validated and successfully applied to urine samples from human subjects orally exposed to monoterpenes trough an intake of cough medication containing monoterpenes as active medicinal ingredients.
Asunto(s)
Alcoholes/orina , Cromatografía Líquida de Alta Presión/métodos , Monoterpenos/orina , Espectrometría de Masa por Ionización de Electrospray/métodos , Cumarinas/química , Combinación de Medicamentos , Colorantes Fluorescentes/química , Humanos , Hidrazinas/química , Mentol/administración & dosificación , Mentol/análogos & derivados , Mentol/metabolismo , Monoterpenos/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Extracción en Fase Sólida/métodosRESUMEN
The metabolism of the bicyclic monoterpene Delta(3)-carene was investigated in vitro using human liver microsomes as well as human smoker/non-smoker lung microsomes and 12 different recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. We detected two metabolites using GC-MS analysis. The mass fragmentation indicated for one metabolite hydroxylation in the allyl position and for the other metabolite epoxidation at the double bond. For clear identification the suggested metabolites were synthesized in a four-step reaction. Comparison of GC retention times and mass spectra lead to the identification of the metabolites as Delta(3)-carene-10-ol ((1S, 6R)-7,7-Dimethylbicyclo[4.1.0]hept-3-en-3-yl-methanol) and Delta(3)-carene-epoxide ((1S, 3S, 5R, 7R)-3,8,8-Trimethyl-4-oxa-tricyclo[5.1.0.0(3,5)]octane). Delta(3)-carene-10-ol was formed by human liver microsomes and recombinant human CYP2B6, CYP2C19 and CYP2D6. Delta(3)-Carene-epoxide was obviously catalyzed only by CYP1A2. In both cases there was a clear correlation between the metabolite formation, incubation time and enzyme concentration, respectively. Further kinetic analysis revealed that CYP2B6 exhibited the highest activity for Delta(3)-carene 10-hydroxylation. Michaelis-Menten K(m) and V(max) for oxidation of Delta(3)-carene were 0.6 mM and 28.4 nmol/min/nmol P450 using human CYP2B6. For the formation of Delta(3)-carene-epoxide 98.2 mM and 3.9 nmol/min/nmol P450 were determined as K(m) and V(max) by using human CYP1A2. To our knowledge, this is the first time that Delta(3)-carene-10-ol and Delta(3)-carene-epoxide are described as human metabolites of Delta(3)-carene.
Asunto(s)
Compuestos Bicíclicos con Puentes/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Monoterpenos/metabolismo , Monoterpenos Bicíclicos , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/genética , Compuestos Epoxi/química , Compuestos Epoxi/aislamiento & purificación , Compuestos Epoxi/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Humanos , Hidroxilación , Pulmón/metabolismo , Metanol/análogos & derivados , Metanol/química , Metanol/aislamiento & purificación , Metanol/metabolismo , Microsomas/metabolismo , Microsomas Hepáticos/metabolismo , Monoterpenos/química , Monoterpenos/aislamiento & purificación , Oxidación-Reducción , Proteínas Recombinantes/metabolismo , FumarRESUMEN
Human metabolism of the monoterpene cyclic ether 1,8-cineole was investigated in vitro and in vivo. In vitro, the biotransformation of 1,8-cineole was investigated by human liver microsomes and by recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. Besides the already described metabolite 2alpha-hydroxy-1,8-cineole we found another metabolite produced at high rates. The structure was identified by a comparison of its mass spectrum and retention time with the reference compounds as 3alpha-hydroxy-1,8-cineole. There was a clear correlation between the concentration of the metabolites, incubation time and enzyme content, respectively. CYP3A4/5 antibody significantly inhibited the 2alpha- and 3alpha-hydroxylation catalyzed by pooled human liver microsomes. Further kinetic analysis revealed that the Michaelis-Menten K(m) and V(max) for oxidation of 1,8-cineole in position three were 19 microM and 64.5 nmol/min/nmol P450 for cytochrome P450 3A4, and 141 microM and 10.9 nmol/min/nmol P450 for cytochrome P450 3A5, respectively. To our knowledge, this is the first time that 3alpha-hydroxy-1,8-cineole is described as a human metabolite of 1,8-cineole. We confirmed these in vitro results by the investigation of human urine after the oral administration of cold medication containing 1,8-cineole. In human urine we found by GC-MS analysis the described metabolites, 2alpha-hydroxy-1,8-cineole and 3alpha-hydroxy-1,8-cineole.