In vivo formation and bioavailability of isothiocyanates from glucosinolates in broccoli as affected by processing conditions.

SCOPE: To study the effect of residual myrosinase (MYR) activity in differently processed broccoli on sulforaphane (SR) and iberin (IB) formation, bioavailability, and excretion in human volunteers. METHODS AND RESULTS: Five different broccoli products were obtained with similar glucoraphanin (GR) and glucoiberin (GI) content, yet different MYR activity. Excretion of SR and IB conjugates in urine were determined in 15 participants after ingestion of the broccoli products. A reduction of 80% of MYR in the product did not cause differences in the total amount of SR and IB found in urine compared to the product with 100% MYR. Complete inactivation of MYR gave the lowest total amount of SR and IB in urine (10 and 19%). A residual MYR of only 2% in the product gave an intermediate amount (17 and 29%). The excretion half-lives of SR and IB conjugates were comparable for all the products (2.5 h on average), although the maximum excretion peak times were clearly shorter when the residual MYR was higher (2.3-6.1 h). CONCLUSION: For the first time, the effect of residual MYR activity on isothiocyanate bioavailability was systematically and quantitatively studied. Processing conditions have a large effect on the kinetics and bioavailability of isothiocyanates from broccoli.

Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya.

Isothiocyanates are electrophiles that are able to induce phase II biotransformation enzyme gene expression via an electrophile-responsive element (EpRE) in the gene regulatory region. To study the potency of different isothiocyanates to induce the expression of EpRE-regulated genes, a Hepa-1c1c7 luciferase reporter cell line was exposed to structurally different isothiocyanates. The reporter cell line, EpRE(mGST-Ya)-LUX, contains the EpRE from the regulatory region of the mouse glutathione S-transferase Ya gene. Isothiocyanates containing a methyl-sulfur side chain, e.g. sulforaphane, showed a lower EC(50) (0.8-3.2 microM) and a comparable induction factor (17-22.4) compared to the structurally different isothiocyanates containing an alkyl or aromatic side chain, e.g. allyl and phenylethyl isothiocyanate (EC(50) 3.9-6.5 microM, induction factor 17.5-23). After 24h of exposure, on average (+/-SD) 23+/-5% of the isothiocyanate was found in the cells and 77% in the cell medium. Isothiocyanates prove to be strong inducers of electrophile-responsive element-mediated gene expression at physiological concentrations. The here described luciferase reporter cell line is a suitable assay to measure the potency of compounds to induce EpRE-regulated gene expression.

Bioavailability and kinetics of sulforaphane in humans after consumption of cooked versus raw broccoli.

The aim of this study was to determine the bioavailability and kinetics of the supposed anticarcinogen sulforaphane, the hydrolysis product of glucoraphanin, from raw and cooked broccoli. Eight men consumed 200 g of crushed broccoli, raw or cooked, with a warm meal in a randomized, free-living, open cross-over trial. Higher amounts of sulforaphane were found in the blood and urine when broccoli was eaten raw (bioavailability of 37%) versus cooked (3.4%, p ) 0.002). Absorption of sulforaphane was delayed when cooked broccoli was consumed (peak plasma time ) 6 h) versus raw broccoli (1.6 h, p ) 0.001). Excretion half-lives were comparable, 2.6 and 2.4 h on average, for raw and cooked broccoli, respectively (p ) 0.5). This study gives complete kinetic data and shows that consumption of raw broccoli results in faster absorption, higher bioavailability, and higher peak plasma amounts of sulforaphane, compared to cooked broccoli.

Association between consumption of cruciferous vegetables and condiments and excretion in urine of isothiocyanate mercapturic acids.

A high intake of cruciferous vegetables is associated with a reduced risk of cancer and cardiovascular diseases. This protective effect has been linked to isothiocyanates, enzymatic hydrolysis products of glucosinolates. In this study, the metabolic fate of glucosinolates and isothiocyanates after ingestion of 19 different cruciferous vegetables was studied in three male subjects. After the consumption of 13 cruciferous vegetables (glucosinolate content, 0.01-0.94 mmol/kg) and six condiments (isothiocyanate content, 0.06-49.3 mmol/kg), eight different isothiocyanate mercapturic acids were determined in urine samples. Excretion levels after the consumption of raw vegetables and condiments were higher (bioavailability, 8.2-113%) as compared to cooked vegetables (bioavailability, 1.8-43%), but the excretion rate was similar (t1/2=2.1-3.9 h). Isothiocyanates in urine remain longer at a nonzero level after the consumption of glucosinolates from cooked vegetables, as compared to raw vegetables and condiments, and maximal levels in urine were reached about 4 h later. Isothiocyanate mercapturic acids can be used as a biomarker to reflect the active dose of isothiocyanates absorbed.

Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity.

The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from the human NAD(P)H:quinone oxidoreductase (hNQO1) and the mouse glutathione-S-transferase Ya (mGST-Ya) gene, containing one and two tandem EpRE core sequences, respectively. The standard inducer tert-butylhydroquinone (tBHQ), the electrophile benzyl isothiocyanate (BITC), and the antioxidant flavonoid quercetin were found to induce luciferase expression, thereby validating these newly developed reporter cell lines. For tBHQ and BITC, but not for quercetin, higher maximum luciferase induction was found under control of the mGST-Ya EpRE as compared to the hNQO1 EpRE, pointing at different induction mechanisms. Furthermore, we investigated the structure-activity relationship for induction of luciferase expression by flavonoids in EpRE(mGST-Ya)-LUX cells, and also the relation between luciferase induction and flavonoid antioxidant potency. Five different flavonoids with a planar molecular structure were found to induce various levels of luciferase activity, whereas taxifolin, a non-planar flavonoid, did not induce luciferase activity. This suggests that a stereospecific molecular interaction may be important for EpRE-mediated gene activation, possibly with Keap1, a regulator of EpRE-controlled transcription, or with another effector or receptor protein. No consistent relation between luciferase induction level and flavonoid antioxidant potential was observed. Altogether, these results point to differences in induction mechanism between the various chemoprotective compounds tested. The newly developed stably transfected reporter cell lines provide a validated tool for future screening and mechanistic studies of EpRE-mediated gene transcription.

Synthesis of isothiocyanate-derived mercapturic acids.

Twelve mercapturic acids derived from saturated and unsaturated aliphatic and aromatic isothiocyanates were synthesised, by adding isothiocyanate to a solution of N-acetyl-L-cysteine and sodium bicarbonate, in a typical yield of 77%. Isothiocyanates were synthesised first by adding the corresponding alkyl bromide to phthalimide potassium salt. The obtained N-alkyl-phthalimide was hydrazinolysed yielding the alkyl amine, which subsequently was reacted with thiophosgene yielding the isothiocyanate with an overall yield of 16%. Mercapturic acids in urine can serve as a biomarker of intake to determine the health promoting potential of isothiocyanates present in cruciferous vegetables.

Analysis of isothiocyanate mercapturic acids in urine: a biomarker for cruciferous vegetable intake.

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.

Metabolism of sinigrin (2-propenyl glucosinolate) by the human colonic microflora in a dynamic in vitro large-intestinal model.

Cruciferous vegetables, such as Brassica, which contain substantial quantities of glucosinolates, have been suggested to possess anticarcinogenic activity. Cutting and chewing of cruciferous vegetables releases the thioglucosidase enzyme myrosinase, which degrades glucosinolates to isothiocyanates and other minor metabolites. Cooking of cruciferous vegetables inactivates the myrosinase enzyme, allowing intact glucosinolates to reach the large intestine, where they can be degraded by the indigenous microflora into isothiocyanates. This local release of isothiocyanates may explain the protective effect of cruciferous vegetables on the colon epithelium. However, little is known about the amounts and identities of glucosinolate metabolites produced by the human microflora. The production of allyl isothiocyanate from sinigrin was investigated in a dynamic in vitro large-intestinal model, after inoculation with a complex microflora of human origin. Sinigrin and allyl isothiocyanate concentrations were analysed in the lumen and dialysis fluid of the model. Peak levels of allyl isothiocyanate were observed between 9 and 12 h after the addition of sinigrin. The model was first set up with a pooled and cultured human microflora, in which 1 and 4% of, respectively, 1 and 15 mM sinigrin, was converted into AITC. However, the conversion rate was remarkably higher if different individual human microflora were used. Between 10% and 30% (mean 19%) of the sinigrin was converted into allyl isothiocyanate. The results of this study suggest that allyl isothiocyanate is converted further into other, yet unknown, metabolites.