Isothiocyanates (ITCs) derived from plants of the order Brassicales are known for their antibacterial, anti-inflammatory or anticarcinogenic potential. Although only the free ITCs exert bioactivity, quantification in vivo is almost exclusively performed on total ITC/metabolite content. We therefore investigated in a pilot study the amount of free ITC at different steps critical for therapeutic efficacy. A sensitive and specific GC-MS/MS method for the simultaneous quantification of individual free ITC after solid-phase extraction (SPE) was developed. We show here that release of biologically active ITC from plants occurs at not only alkaline but also acidic pH. Furthermore, in human urine conversion of the ultimate, inactive mercapturic acid conjugate back into its corresponding bioactive form is increased at alkaline as compared to neutral pH. This was also observed in the urine of human volunteers, where - in correlation with the pH value - a mean of 0.16 to 1.03 μmol ITC was detected after oral application of a phytotherapeutic agent containing 30.4 μmol of the initial pro-drugs. The amounts of free ITC being necessary for bioactivity in vitro were found to be indeed achieved in vivo. These data might be helpful to better understand the beneficial effects of ITC observed in vivo.
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