Analysis of glucosinolates from broccoli and other cruciferous vegetables by hydrophilic interaction liquid chromatography.

While methods for the identification and quantification of total plant glucosinolate content typically utilize desulfation of glucosinolates followed by reversed-phase chromatography, the analysis of intact glucosinolates has been problematic. Hydrophilic interaction chromatography offers a novel method for analyzing intact glucosinolates and when performed along with ion-pair reversed-phase chromatography offers a powerful and complementary method for glucosinolate analysis.

Using isothiocyanate excretion as a biological marker of Brassica vegetable consumption in epidemiological studies: evaluating the sources of variability.

OBJECTIVE: Brassica vegetable consumption (e.g. broccoli) leads to excretion of isothiocyanates (ITC) in urine. We evaluated the consistency of ITC as a biomarker for dietary Brassica vegetable consumption across the types of vegetables and methods of preparation used in Western societies, and across consumption levels. DESIGN: A single-armed behavioural intervention with duplicate baseline assessment and post-intervention assessment. Urinary ITC excretion and estrogen metabolites were measured from 24-hour urine samples. Dietary intake was measured by a 24-hour recall. SETTING: The behavioural intervention facilitated daily Brassica intake among participants by providing peer support, food preparation instruction, guided practice in a teaching kitchen, and other information. SUBJECTS: Thirty-four healthy free-living postmenopausal women who recently had a negative screening mammogram at the University of Massachusetts Medical Center. RESULTS: Urinary ITC excretion and total Brassica intake followed the same pattern over the intervention. The ITC biomarker significantly predicted Brassica intake when Brassica consumption averaged about 100 g day-1, but not when Brassica consumption averaged about 200 g day-1. Urinary ITC levels were somewhat higher when more raw vegetables were consumed as compared to lightly cooked vegetables, while the types of Brassica consumed appeared to have only a small, non-significant effect on urinary ITC levels. CONCLUSION: Urinary ITC excretion would be a good exposure biomarker among populations regularly consuming a vegetable serving/day, but may be less accurate among populations with greater intake levels or a wide range of cooking practices.

Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans.

Broccoli sprouts are a rich source of glucosinolates and isothiocyanates that induce phase 2 detoxication enzymes, boost antioxidant status, and protect animals against chemically induced cancer. Glucosinolates are hydrolyzed by myrosinase (an enzyme found in plants and bowel microflora) to form isothiocyanates. In vivo, isothiocyanates are conjugated with glutathione and then sequentially metabolized to mercapturic acids. These metabolites are collectively designated dithiocarbamates. We studied the disposition of broccoli sprout glucosinolates and isothiocyanates in healthy volunteers. Broccoli sprouts were grown, processed, and analyzed for (a) inducer potency; (b) glucosinolate and isothiocyanate concentrations; (c) glucosinolate profiles; and (d) myrosinase activity. Dosing preparations included uncooked fresh sprouts (with active myrosinase) as well as homogenates of boiled sprouts that were devoid of myrosinase activity and contained either glucosinolates only or isothiocyanates only. In a crossover study, urinary dithiocarbamate excretion increased sharply after administration of broccoli sprout glucosinolates or isothiocyanates. Cumulative excretion of dithiocarbamates following 111-micromol doses of isothiocyanates was greater than that after glucosinolates (88.9 +/- 5.5 and 13.1 +/- 1.9 micromol, respectively; P < 0.0003). In subjects fed four repeated 50-micromol doses of isothiocyanates, the intra- and intersubject variation in dithiocarbamate excretion was very small (coefficient of variation, 9%), and after escalating doses, excretion was linear over a 25- to 200-micromol dose range. Dithiocarbamate excretion was higher when intact sprouts were chewed thoroughly rather than swallowed whole (42.4 +/- 7.5 and 28.8 +/- 2.6 micromol; P = 0.049). These studies indicate that isothiocyanates are about six times more bioavailable than glucosinolates, which must first be hydrolyzed. Thorough chewing of fresh sprouts exposes the glucosinolates to plant myrosinase and significantly increases dithiocarbamate excretion. These findings will assist in the design of dosing regimens for clinical studies of broccoli sprout efficacy.

Human metabolism and excretion of cancer chemoprotective glucosinolates and isothiocyanates of cruciferous vegetables.

Isothiocyanates and their naturally occurring glucosinolate precursors are widely consumed as part of a diet rich in cruciferous vegetables. When plant cells are damaged, glucosinolates are released and converted to isothiocyanates by the enzyme myrosinase. Many isothiocyanates inhibit the neoplastic effects of various carcinogens at a number of organ sites. Consequently, these agents are attracting attention as potential chemoprotectors against cancer. As a prerequisite to understanding the mechanism of the protective effects of these compounds, which is thought to involve the modulation of carcinogen metabolism by the induction of phase 2 detoxication enzymes and the inhibition of phase 1 carcinogen-activating enzymes, we examined the fate of ingested isothiocyanates and glucosinolates in humans. Recently developed novel methods for quantifying isothiocyanates (and glucosinolates after their quantitative conversion to isothiocyanates by purified myrosinase) and their urinary metabolites (largely dithiocarbamates) have made possible a detailed examination of the fates of isothiocyanates and glucosinolates of dietary crucifers. In a series of studies in normal volunteers, we made these findings. First, in nonsmokers, urinary dithiocarbamates were detected only after the consumption of cruciferous vegetables and condiments rich in isothiocyanates and/or glucosinolates. In sharp contrast, the consumption of noncrucifers (corn, tomatoes, green beans, and carrots) did not lead to the excretion of dithiocarbamates. Moreover, the quantities of dithiocarbamates excreted were related to the glucosinolate/isothiocyanate profiles of the cruciferous vegetables administered (kale, broccoli, green cabbage, and turnip roots). Second, eating prepared horseradish containing graded doses of isothiocyanates (12.3-74 micromol; mostly allyl isothiocyanate) led to a rapid excretion of proportionate amounts (42-44%) of urinary dithiocarbamates with first-order kinetics. The ingestion of broccoli in which myrosinase had been heat-inactivated also led to proportionate but low (10-20%) recoveries of urinary dithiocarbamates. Broccoli samples subsequently treated with myrosinase to produce the cognate isothiocyanates were much more completely (47%) converted to dithiocarbamates. Finally, when bowel microflora were reduced by mechanical cleansing and antibiotics, the conversion of glucosinolates became negligible. These results establish that humans convert substantial amounts of isothiocyanates and glucosinolates to urinary dithiocarbamates that can be easily quantified, thus paving the way for meaningful studies of phase 2 enzyme induction in humans.