Analysis of Isothiocyanate Mercapturic Acids in Urine:  A Biomarker for Cruciferous Vegetable Intake

Abstract

Cruciferous vegetables contain glucosinolates, which are degraded to isothiocyanates. These are easily absorbed, conjugated to glutathione, and excreted into the urine as their corresponding mercapturic acids. We have developed and validated a solid phase extraction-high-performance liquid chromatography-electrospray ionization mass spectrometry/mass spectrometry method for the specific analysis of individual isothiocyanate mercapturic acids in urine. The range of reliable analysis was 1.0-310 microM in urine. Urine samples fortified with three different levels of isothiocyanate mercapturic acids were measured on six different days by three independent technicians. The relative standard deviation (RSD) of repeatability was 12, 6, and 3%; the RSD of reproducibility was 19, 14, and 8%, and spike recoveries were 103, 104, and 103%, respectively, for 1.04, 10.5, and 313 microM levels. In 24 h urine collected from two volunteers after they consumed broccoli and cauliflower, clearly sulforaphane mercapturic acid (133 micromol) and allyl isothiocyanate mercapturic acid (4.7 micromol) were found. This procedure demonstrates a reliable and efficient method to study the intake and mode of action of isothiocyanates in animal studies and clinical trials.

Antiproliferative activity of sulforaphane in Akt-overexpressing ovarian cancer cells

“…High levels of sulforaphane have been reported in mammalian cells and up to 20 Amol/L in rat plasma (30) and in urine of humans consuming broccoli (31,32), suggesting that sulforaphane is bioavailable. The mean glucosinolates content is 100 mmol/L per 100 g of fresh weight corresponding to 26 mg glucoraphanin accounting for 55% of the glucosinolates (33).…”

Purification of major glucosinolates from Brassicaceae seeds and preparation of isothiocyanate and amine metabolites

“…36,37 No method has been published for the preparation and analysis of amine derivatives. Four of the six amine degradation products are not available commercially and were prepared by organic synthesis: amine derivatives that are putative metabolites of gluconapin, glucoiberin, glucoraphanin and glucoalyssin.…”

Improved synthesis methods of standards used for quantitative determination of total isothiocyanates from broccoli in human urine

“…In humans ITCs are primarily metabolised via the mercapturic acid pathway to the N-acetylcystein conjugate of ITC, and this metabolite is excreted in urine and could thereby serve as a marker for absorbed ITC [7,8]. In this present investigation, we have chosen to look at total ITC excretion as a biomarker of overall ITC exposure.…”

“…In this present investigation, we have chosen to look at total ITC excretion as a biomarker of overall ITC exposure. Previous studies [8,9] have investigated how individual ITC metabolites can be reliably quantified and used as biomarker for exposure for individual glucosinolates. However, precaution must be taken when assuming that the excreted ITC conjugates are equivalent to the consumed amounts of ITCs, since ITC conjugates are unstable and rapidly dissociate back to ITCs [10].…”