Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli.

The cancer-chemopreventive effects of broccoli may be attributed, in part, to isothiocyanates (ITCs), hydrolysis products of glucosinolates. Glucosinolates are hydrolyzed to their respective ITCs by the enzyme myrosinase, which is inactivated by heat. In this study, the metabolic fate of glucosinolates after ingestion of steamed and fresh broccoli was compared in 12 male subjects in a crossover design. During each 48-hour baseline period, no foods containing glucosinolates or ITCs were allowed. The subjects then consumed 200 g of fresh or steamed broccoli; all other dietary sources of ITCs were excluded. Blood and urine samples were collected during the 24-hour period after broccoli consumption. Total ITC equivalents in broccoli and total ITC equivalents in plasma and urine were assayed by high-performance liquid chromatography as the cyclocondensation product of 1,2-benzenedithiol. The content of ITCs in fresh and steamed broccoli after myrosinase treatment was found to be virtually identical (1.1 vs. 1.0 micromol/g wet wt). The average 24-hour urinary excretion of ITC equivalents amounted to 32.3 +/- 12.7% and 10.2 +/- 5.9% of the amounts ingested for fresh and steamed broccoli, respectively. Approximately 40% of total ITC equivalents in urine, 25.8 +/- 13.9 and 6.9 +/- 2.5 micromol for fresh and steamed broccoli, respectively, occurred as the N-acetyl-L-cysteine conjugate of sulforaphane (SFN-NAC). Total ITC metabolites in plasma peaked between 0 and 8 hours, whereas urinary excretion of total ITC equivalents and SFN-NAC occurred primarily between 2 and 12 hours. Results of this study indicate that the bioavailability of ITCs from fresh broccoli is approximately three times greater than that from cooked broccoli, in which myrosinase is inactivated. Considering the cancer-chemopreventive potential of ITCs, cooking broccoli may markedly reduce its beneficial effects on health.

Conversion of glucosinolates to isothiocyanates in humans after ingestion of cooked watercress.

Isothiocyanates (ITCs), major constituents of cruciferous vegetables, can inhibit tumorigenesis in rodents by modulating the metabolism of carcinogens. ITCs that occur as glucosinolates are released by myrosinase-mediated hydrolysis when raw vegetables are chopped or chewed. However, because cruciferous vegetables are commonly consumed by humans after being cooked, it is important to examine whether dietary glucosinolates are converted to ITCs after cooked cruciferous vegetables in which myrosinase is deactivated have been consumed. This information is useful for evaluating the potential role of ITCs in cruciferous vegetables in the protection against human cancers. A urinary marker, based on a cyclocondensation product formed by the reaction of ITCs and their conjugates with 1,2-benzenedithiol, was used to quantify the uptake of dietary ITCs in humans. At breakfast and lunch, nine volunteers consumed a total of 350 g of cooked watercress in which the myrosinase activity was completely deactivated. On the basis of the analysis of ITCs in the cooked watercress upon adding exogenous myrosinase, the amount of glucosinolates ingested by each subject was estimated to be 475 micromol. The 24-h urine samples showed that the total urinary excretion of ITC conjugates in the subjects ranged from 5.6 to 34.8 micromol, corresponding to 1.2-7.3% of the total amount ingested. On the basis of our previous results that approximately 50% of dietary ITCs were excreted in the urine as conjugates, these values represent the minimal in vivo conversion of glucosinolates to ITCs. For purposes of comparison, we carried out a second experiment in which 150 g of uncooked watercress were consumed. The percentage of urinary ITC conjugates excreted in this study ranged from 17.2 to 77.7% of the total ingested ITCs. These results indicate that glucosinolates are converted to ITCs in humans after ingestion of cooked watercress, in which the myrosinase has been completely inactivated. The extent of conversion, however, is considerably less than that after ingesting uncooked vegetables. Furthermore, upon incubation of the cooked watercress juice with fresh human feces under anaerobic conditions, approximately 18% of glucosinolates was hydrolyzed to ITCs in 2 h. These results suggest that the microflora in the intestinal tract are a likely source for the hydrolysis of glucosinolates to ITCs in humans.

A urinary biomarker for uptake of dietary isothiocyanates in humans.

Isothiocyanates (ITCs) are a family of biologically active compounds that are distributed widely in cruciferous vegetables. Although studies in rodents have shown that these compounds are effective and versatile inhibitors of tumorigenesis, the role of dietary ITCs in the protection against human cancers remains to be established. A prerequisite of human studies is to develop an uptake biomarker for dietary ITCs. In this study, we describe a rapid high-performance liquid chromatography-based assay to measure the total ITC level in human urine. This assay is based on a previously described reaction of ITCs or their thiol conjugates with 1,2-benzenedithiol to yield a cyclocondensation product, 1,3-benzodithiole-2-thione, which then can be quantified by reverse phase high-performance liquid chromatography with UV detection. This new assay was validated by analyzing urine samples from 14 subjects who had consumed a known amount of watercress or brown mustard in a controlled experiment. The N-acetylcysteine conjugates of phenethyl ITC and allyl ITC from watercress and brown mustard, respectively, were quantified and compared with the results obtained from the current assay. Results of the two methods were highly correlated (r = 0.978), indicating the specificity of this new assay for dietary ITCs. The feasibility of this assay for population-based studies was examined using stored urine samples collected from nine participants of a prospective cohort study in Shanghai, China, who indicated that they were daily consumers of dark green vegetables. There was a 10-fold variation in urinary ITC contents among these samples, ranging from 0.7 to 7.0 micromol/g creatinine. These results show the potential use of this uptake biomarker in epidemiological studies to identify the role of dietary ITCs in modifying cancer risks in humans.